Lipidomic analysis of the retina in a rat model of Smith–Lemli–Opitz syndrome: alterations in docosahexaenoic acid content of phospholipid molecular species

Lipidomic Analysis of the Retina in a Rat Model of Smith-Lemli-Opitz Syndrome: Alterations in Docosahexaenoic Acid Content ofPhospholipid Molecular Species

David A. Ford1, Julie K. Monda1, Richard S. Brush2, Robert E. Anderson2,3, Michael J.Richards4, and Steven J. Fliesler4,5,*1E. A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School ofMedicine, Saint Louis, MO 63104 U.S.A.2Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City,OK 73104 U.S.A.3Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK73104 U.S.A.4Department of Ophthalmology, Saint Louis University School of Medicine, Saint Louis, MO 63104U.S.A.5Department of Pharmacological & Physiological Science, Saint Louis University School ofMedicine, Saint Louis, MO 63104 U.S.A.

AbstractSmith-Lemli-Opitz syndrome (SLOS) is a complex hereditary disease caused by an enzymatic defectin the last step of cholesterol biosynthesis. Progressive retinal degeneration occurs in an AY9944-induced rat model of SLOS, with biochemical and electroretinographic hallmarks comparable to thehuman disease. We evaluated alterations in the non-sterol lipid components of the retina in this model,compared to age-matched controls, using lipidomic analysis. The levels of 16:0–22:6 and 18:0–22:6phosphatidylcholine molecular species in retinas were less by >50% and >33%, respectively, in ratstreated for either two or three months with AY9944. Relative to controls, AY9944 treatment resultedin >60% less di-22:6 and >15% less 18:0–22:6 phosphatidylethanolamine molecular species. Thepredominant phosphatidylserine molecular species in control retinas were 18:0–22:6 and di-22:6;notably, AY9944 treatment resulted in >80% less di-22:6 phosphatidylserine, relative to controls.Remarkably, these changes occurred in the absence of n3 fatty acid deficiency in plasma or liver.Thus, the retinal lipidome is globally altered in the SLOS rat model, relative to control rats, with themost profound changes being less phosphatidylcholine, phosphatidylethanolamine, andphosphatidylserine molecular species containing docosahexaenoic acid (22:6). These findingssuggest that SLOS may involve additional metabolic compromise beyond the primary enzymaticdefect in the cholesterol pathway.

KeywordsDocosahexaenoic acid; fatty acid; lipidomic analysis; retina; AY9944

*Address correspondence to: Dr. Steven J. Fliesler, Saint Louis University Eye Institute, 1755 South Grand Blvd.- ABI 506, Saint Louis,MO 63104 U.S.A. Tel: (314)256-3252; Fax: (314)771-2317; Email: E-mail: [email protected].

NIH Public AccessAuthor ManuscriptJ Neurochem. Author manuscript; available in PMC 2009 August 6.

Published in final edited form as:J Neurochem. 2008 May ; 105(3): 1032–1047. doi:10.1111/j.1471-4159.2007.05203.x.

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Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive, multiple congenitalanomalies disorder caused by a defect in the last enzymatic step in the cholesterol biosyntheticpathway (Smith et al. 1964) (reviewed in: Yu and Patel 2005; Correa-Cerro and Porter 2005;Kelley and Hermann 2001). The dyslipidemia in patients affected with this disorder has aspecific biochemical signature: blood and other tissues contain abnormal (typically grosslyelevated) levels of 7-dehydrocholesterol (7DHC) and markedly reduced levels of cholesterol(Chol), relative to the levels found in normal controls (Irons et al. 1993; Tint et al. 1994). Thisis due to mutations in the DHCR7 gene, which encodes the enzyme 3β-hydroxy-sterol-Δ7-reductase (EC 1.3.1.21) (Yu and Patel 2005; Correa-Cerro and Porter 2005; Waterham et al.1998; Fitzky et al. 1998), thereby altering the efficiency of conversion of 7DHC to Chol. Thenervous system, in particular, is profoundly affected in this disease, as manifested by cognitivefunction deficits (e.g., mental retardation, autism) (Nowaczyk et al. 1999; Tierney et al.2000; Sikora et al. 2006). In addition, retinal dysfunction has been reported in SLOS patients,particularly with regard to rod photoreceptor electrophysiology (Elias et al. 2003). An animalmodel of this human disease has been developed by treating rats with AY9944, a selectivepharmacological inhibitor of the same enzyme that is defective in SLOS (Kolf-Klauw et al.1996; Wolf et al. 1996; Dvornik et al. 1963; Givner and Dvornik 1965). Using a modificationof the originally developed model that allows long-term postnatal survival, a progressive retinaldegeneration affecting both rod and cone photoreceptors has been described (Fliesler et al.2004). Whereas the normal rat retina contains virtually no 7DHC, the mole ratio of 7DHC/Chol in retinas of one-month old rats treated with AY9944 since gestation approaches ca. 4:1,but without associated histological or functional impairments (Fliesler et al. 1999). In contrast,by three postnatal months, the 7DHC/Chol ratio in retinas of AY9944-treated rats reaches levels>5:1, with marked histological degeneration and associated electrophysiological defects(Fliesler et al. 2004). Recently, it was shown that feeding a highcholesterol diet to these ratsfrom weaning through three postnatal months provides a marked improvement primarily incone photoreceptor function, and also significantly improves (toward normal) the sterol profileof the retina, but does not spare the retina from degeneration (Fliesler et al. 2007).

In the course of our studies of the retinal degeneration in the SLOS rat model, we examinedthe lipidomic profile of the retina as a function of postnatal age, in comparison with age-matched control retinas, to assess possible changes in lipid species other than sterols. We reportherein that the fatty acid composition of the major retinal phospholipid molecular species isdramatically altered in the SLOS rat model, relative to that of controls. Remarkably, the relativedocosahexaenoic acid (DHA; 22:6n3) content of these phospholipids was significantly reducedwithin a three-month time course, to an extent comparable to or greater than that achievedheretofore by raising rats on a n3 fatty acid-deficient diet (Futterman et al. 1971; Andersonand Maude 1972; Tinoco et al. 1977; Wiegand et al. 1991; Anderson et al. 1992; Bush et al.1994), except under extraordinary circumstances (Tinoco et al. 1978; Ward et al. 1996;Moriguchi et al. 2004). However, under the conditions employed in this study, there was noevidence of generalized, systemic DHA or n3 fatty acid deficiency. We discuss these findingswithin the context of the role of lipids in supporting normal visual function, and also withregard to the pathobiology of SLOS. To the extent that the AY9944-induced rat model faithfullymimics the human disease, these findings suggest that the metabolic defect in SLOS mayinvolve other pathways beyond the primary defect in the cholesterol pathway.

EXPERIMENTAL PROCEDURESMaterials

Authentic glycerophospholipid standards were obtained from Avanti Polar Lipids, Inc(Alabaster, AL). Fatty acid standards were used as purchased from (Nu-Chek Prep, Inc.(Elysian, MN). Sterol standards were obtained from Steraloids, Inc. (Newport, RI); 7DHC was

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periodically recrystallized from methanol-water and its purity verified by HPLC prior to use.AY9944 (trans-l,4-bis(2-chlorobenzylamino-methyl)cyclohexane dihydrochloride) wascustom synthesized, and matched the spectroscopic and physical properties of an authenticsample of AY9944 (kindly provided by Wyeth-Ayerst Laboratories). All organic solvents wereof HPLC grade, and used as purchased from Fisher Scientific (Pittsburgh, PA). Unlessotherwise specified, all other reagents were used as purchased from Sigma/Aldrich (St. Louis,MO).

AnimalsPregnant Sprague-Dawley rats (6 days sperm-positive) were obtained from Harlan(Indianapolis, IN). Rats (both control and treated) were fed water and a standard rat chow(Purina Mills TestDiet, Richmond, IN), ad lib. The levels of cholesterol in this chow werebelow the detectable limit (10 ppm; S.J. Fliesler, data not shown). Rats were treated withAY9944 as previously described (Fliesler et al. 2004; Fliesler et al. 2007). In brief, pregnantrats implanted with subcutaneous Alzet® osmotic pumps (Model 2ML4; Durect Corporation,Cupertino, CA) containing a PBS solution of AY9944 (1.5 mg/mL), so as to deliver the drugat a constant rate (0.37mg/kg/day, at 2.5 µL/h) from gestational day 7 through the secondpostnatal week. Control dams received the same food and water ad lib, but were given no othertreatment. Pups from AY9944-treated dams were injected subcutaneously three times perweek, on alternating days, with AY9944 (30 mg/kg, in PBS), starting at postnatal day one (P1)and continuing throughout life. Control pups were not treated with any injections, since priorstudies (unpublished results; cf. Fliesler et al. 1999) showed no effect of parallel vehicleinjections with regard to tissue biochemistry, histology, or retinal function. All proceduresinvolving animals were approved by the Saint Louis University IACUC and conformed to theNational Institutes of Health’s Guide for the Care and Use of Laboratory Animals and theAssociation for Research in Vision and Ophthalmology’s Statement for the Use of Animals inOphthalmic and Visual Research.

Retinal lipid extractionWhole retinas were harvested from rats in each group (experimental and control) and wereimmediately frozen in liquid nitrogen in the presence of argon-purged, chilled PBS containingDTPA (0.1 mol/L, BHT (0.01 mg/mL), and SnCl2 (0.01 mg/mL). Retinas were subsequentlyextracted by a modified Bligh-Dyer technique (Ward et al. 1996) in the presence of internalstandards. In brief, lipids from one retina were extracted in a Teflon/glass homogenizer using2 mL of methanol/chloroform (1:1, by vol.) with phase separation by the addition of 1.5 mLof saline. The methanol/chloroform mixture contained the following internal standards: di-14:0phosphatidylethanolamine (PE; 23.6 nmol), di-17:0 PE (23.6 nmol) di-20:0phosphatidylcholine (PC; 11.8 nmol), and di-14:0 phosphatidylserine (PS; 1.8 nmol). Lipidswere extracted twice from the retinas and the pooled chloroform layers were washed once with0.9% (w/v) saline prior to evaporation of solvent under a nitrogen stream and resuspended inchloroform. Lipid extracts were stored under argon at −85 °C in darkness until ready foranalysis.

Serum and liver lipid extraction and fatty acid analysisWhole blood was collected by cardiac puncture from deeply anesthetized animals, and serumwas prepared there from by centrifugation, aliquoted into polypropylene microfuge tubes, flashfrozen in liquid nitrogen, and stored at −85 °C until ready for analysis. Samples were protectedfrom exposure to light as much as possible throughout processing. After thawing, a mixturecontaining known amounts of pentadecanoic (15:0), heptadecanoic (17:0), and heneicosanoic(21:0) acids was added to each sample as internal standards, and total lipids were extractedessentially per the Bligh-Dyer method (Bligh and Dyer 1959), with the minor modifications

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as described previously by Martin et al. (2005). Lipid extracts were dried under a stream ofnitrogen and fatty acids were derivatized to form the corresponding methyl esters (FAMEs),prior to analysis by gas-liquid chromatography (GLC), essentially as described previously(Martin et al. 2005), with the following modifications. In brief, toluene (0.20 mL) plus 1 mlof 2% (by vol.) methanolic sulfuric acid were added to each lipid extract, the mixture wassealed in a glass tube under nitrogen atmosphere with teflon-lined caps, vortexed, and heatedfor 1 h at 100 °C. After cooling on ice, 1.2 mL of water was added, and the FAMEs wereextracted three times with 2.4 mL of hexane, then dried under nitrogen and dissolved in 20 µLnonane. Fatty acid composition was then determined by injecting 3 µL of each sample onto aDB-225 capillary column (30 m x 0.32 mm I.D.; J&W Scientific, Folsom, CA), using anAgilent 6890N gas chromatograph (GC) with model 7683 autosampler (Agilent Technologies,Wilmington, DE), at an inlet temperature of 250 °C and a split ratio of 25:1. The columntemperature was programmed to begin at 160 °C, ramped to 220 °C at 1.33 °C/min, and heldat 220 °C for 18 min. Hydrogen carrier gas flowed at 1.6 mL/min and the flame ionizationdetector temperature was set to 270 °C. The chromatographic peaks were integrated andprocessed with ChemStation® software (Agilent Technologies). FAMES were identified bycomparison of their relative retention times with authentic standards and relative molepercentages were calculated.

Total lipids of liver (50 mg wet wt. specimens) were extracted per the method of Folch et al.(1957). The tissues were homogenized in 4 ml chloroform:methanol (2:1, by vol.). Proteins inthe homogenate were pelleted by centrifugation at 1,000g for 10 min and the lipid extractremoved. The pellet was washed twice with 1 mL chloroform/methanol (1:1, by vol.) and thewashes were combined with the initial lipid extract. The combined lipid extract was thenwashed with 0.2 volumes of 1 mmol/L aqueous DTPA followed by Folch theoretical upperphase (chloroform/methanol/water, 3:48:47, by vol.), each time discarding the aqueous upperphase. The resulting purified lipid extract was dried under a stream of nitrogen and re-suspended in 0.5 mL of toluene. A 0.1 mL aliquot was taken for derivatization and GLC analysisas described above for serum fatty acid analysis.

Electrospray ionization-mass spectrometry (ESI-MS)Extracted retinal lipids were resuspended in methanol/chloroform (4:1, by vol.) to a dilutionof 20 pmol of lipid per µL and analyzed by ESI-MS in the direct infusion mode at a flow rateof 1–3 µL/min using a Thermo Electron TSQ Quantrum Ultra® instrument (Williams et al.2000). Under selected conditions, 10 pmol of NaOH per µL was added to samples immediatelyprior to injection (unless indicated otherwise) and samples were run either in the positive ornegative ion mode. In the positive ion mode, electrospray needle voltage was 4 kV, andcapillary temperature was 280 °C. In the negative ion mode, electrospray needle voltage was3.2 kV, and capillary temperature was 250 °C. Tandem mass spectrometry was performed onselected ions (typical collision energies were ∼28–35 eV) and spectra were averaged over 3–5 min and processed utilizing Xcalibur® (Thermo Electron) software. For cholineglycerophospholipids (PC and corresponding plasmalogens), neutral loss (NL) scanning of59.1 and 183.1 amu was monitored at collision energies of −28 and −32 eV, respectively.Precursor ion scanning for PE (m/z 196), arachidonic acid (20:4; m/z 303.3), stearic acid (18:0;m/z 283.2) and DHA (22:6; m/z 327.3) was performed in the negative ion mode at collisionenergies of 50 eV (for PE) and 35 eV (for fatty acids). Also in the negative ion mode, NLscanning for 87 amu (for PS) and precursor ion scanning for m/z 153 was monitored at collisionenergies of 25 and 35 eV, respectively. Spectra were averaged over 3–5 min and processedutilizing Xcalibur® software (Thermo Electron). Individual molecular species were quantifiedby comparing the ion intensity of individual molecular species to that of the appropriate internalstandards following corrections for type I and type II 13C isotope effects (Han and Gross2005). Values are expressed as the mass of each individual molecular species per retina.

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LC/MS analyses—Retinal lipid extracts were separated on a Thermo Finnigan Surveyor LCequipped with a Hypersil® 150 mm x 1 mm silica column (Thermo) equilibrated with mobilephase A (hexane/isopropanol/1 mol/L ammonium acetate in water, 30/40/2, by vol.) at a flowrate of 60 µL/min. Following injection of lipid extracts onto the column, the column was elutedfor 6 min with mobile phase A followed by a linear gradient to mobile phase B (hexane/isopropanol/1 mol/L ammonium acetate in water; 30/40/7, by vol.) over 9 min. The stationaryphase was further eluted for another 25 min with mobile phase A. Lipids eluted from the columnwere monitored using a TSQ Quantum Ultra triple quadrupole mass spectrometer in thenegative ion mode (electrospray needle voltage, 4 kV; capillary temperature, 275 °C). Lipidswere detected as negative ions as well as through MS/MS techniques, including precursor ionscanning for m/z 196 as well as neutral loss scanning for both 87 amu (for PS) and 74 amu (forPC: loss of methyl acetate from the [M+ H3CCOO]−).

Sterol analysisWhole blood was collected from animals at one and three postnatal months, allowed to clot (at4 °C, in darkness), and serum was prepared therefrom by centrifugation. Sterol compositionwas analyzed by reverse-phase HPLC, after saponification and extraction of thenonsaponifiable lipids, as previously described (Fliesler et al. 1999; Fliesler et al. 1993). Inbrief, each specimen (50 µL total, plus an internal standard of [3H]Chol) was saponified inmethanolic KOH, and the nonsaponifiable lipids were extracted with petroleum ether,redissoved in methanol, and analyzed by reverse-phase HPLC (detection at 205 nm).Identification of sterols was performed in comparison with authentic standards of 7DHC andChol; integrated peak areas were analyzed with respect to empirically determined responsefactors for each sterol, with calculated masses corrected for recovery efficiency, based uponthe recovery of [3H]cholesterol).

Statistical analysesFor the ESI-MS studies, statistical analysis of the data was performed with respect to the effectof AY9944 treatment vs. controls for a given molecular species at the same age (e.g., levels ofdi-22:6 PS at two months postnatal, treated vs. control rats), using a two-tailed Student’s t-testwith a maximum cutoff for statistical significance of P < 0.05. For serum fatty acid analysesperformed by GLC, multivariant ANOVA with post-hoc Scheffe tests were used to determinestatistical significance, with a maximum cutoff of P < 0.05. For the liver fatty acid analyses,statistical significance was evaluated using a two-tailed Student’s t-test, with a maximum cutoffof P < 0.05.

RESULTSGlobal Disruption of Sterol Biosynthesis by AY9944

To verify that AY9944-treated animals exhibited the expected alteration of sterol metabolismconsistent with the biochemical hallmarks of SLOS (i.e., accumulation of 7DHC and reductionin levels of Chol), serum samples collected at one and three postnatal months were analyzedquantitatively for sterol content by reverse-phase HPLC (see Table 1). Serum from AY9944-treated rats exhibited a ca. 2.3-fold increase in the 7DHC/Chol mole ratio as a function oftreatment time: at one month, the ratio was 2.0 ± 0.8, while at three months, the ratio was 4.6± 1.0. Serum from control rats had undetectable levels of 7DHC. Also, in agreement with thefact that AY9944 is a potent hypocholesterolemic drug (Kolf-Klauw et al. 1996;Wolf et al.1996;Dvornik et al. 1963;Givner and Dvornik 1965), the total sterol content of serum at oneand three months in AY9944-treated rats was 16.5 ± 6.2 mg/dL and 20.8 ± 5.6 mg/dL,respectively, compared to 100.5 ± 10.7 mg/dL and 95.5 ± 17.5 in serum samples from controlrats at one and three postnatal months, respectively. On average, AY9944 treatment caused aca. five-fold reduction in total serum sterol levels, compared to controls, over the specified

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treatment period. Hence, under the conditions employed, AY9944-treated animals faithfullymimicked the signature biochemical hallmarks of SLOS, in good agreement with prior studies(Kolf-Klauw et al. 1996;Wolf et al. 1996;Dvornik et al. 1963;Givner and Dvornik1965;Fliesler et al. 2004;Fliesler et al. 1999).

Shotgun Lipidomic Analysis of Phospholipids from Normal Adult Rat RetinaAs a prelude to a detailed comparative analysis of the retinal phospholipid composition ofAY9944-treated vs. age-matched control rats, we used a shotgun lipidomics approach (Hanand Gross 2005) to examine phospholipid composition in the adult, normal rat retina. Figure1 shows the negative ion spectra of retinal phospholipids from a two-month old normalSprague-Dawley rat, using the crude total lipid extract (dissolved in methanol/chloroform, 4:1,by vol.), directly infused into the ESI source in the negative ion mode. Analyses of the massspectrum of the total ion current (TIC) revealed that the predominant molecular speciesobserved under these conditions are those arising from PS and phosphatidylinositol molecularspecies. The PS molecular species were specifically detected by neutral loss (NL) scanning of87 amu (Fig. 1). Precursor ion scanning of m/z 153 showed all species containing a glycerolphosphate moiety, which includes not only PS species, but also phosphatidylinositol molecularspecies. There were three predominant PS molecular species, containing the following diacylpairs (with values for relative mol% of total PS pool given in parentheses): 18:0–20:4 (7.0%),18:0–22:6 (56.8%), and di-22:6 (26.5%), as determined from ions observed at m/z 810.5, 834.5and 878.5, respectively. The molecular ion at m/z 885.5 (top panel, Fig. 1) corresponds to 18:0–20:4 phosphatidylinositol, which forms a strong negative ion in the full negative ion spectrain comparison to the weakly anionic PE molecular species. Because we were interestedprimarily in the three major glycerophospholipid classes (PC, PE, and PS), we did not includean internal phosphatidylinositol standard in the samples. Hence, one cannot reliably quantifythe relative mole percentage of phosphatidylinositols based upon the peak intensities shownin Fig. 1. Prior studies (reviewed in Fliesler and Anderson 1983) indicate thatphosphatidylinositol accounts for only ca. 4% of the total phospholipids in rat retina.Additionally, precursor ion scanning of m/z 283.2, m/z 303.3 and m/z 327.4 (Fig. 1) was usedto identify the individual esterified fatty acids in these phospholipids, which correspond tostearic acid (18:0), arachidonic acid (20:4), and docosahexaenoic acid (DHA, 22:6),respectively.

Figure 2 (top spectrum) shows the PE molecular species, which are observed in the massspectrum from the TIC in negative ion mode following the addition of 10 pmol NaOH per µlof the crude lipid extract. precursor ion scanning of m/z 196 confirmed the presence of PEmolecular species in these spectra and precursor ion scanning following acid treatmentdemonstrated the presence of the acid-labile plasmalogen molecular species (Fig. 2, thirdspectrum from top). The plasmalogen molecular species include 18:1–20:4, 18:0–20:4, and18:0–22:6, with ions at m/z 748.4, 750.5 and 774.5, respectively. We estimate that PEplasmalogens account for 34.8 mol% of the total PE pool in rat retina. The predominant diacylPE molecular species were 18:0–20:4, 18:0–22:6 and di-22:6 with ions at m/z 766.5, 790.4 and834.4, respectively. It should be appreciated from these spectra in the negative ion mode thatsome isobaric species exist that span phospholipid classes. In particular, 18:0–22:6 PS anddi-22:6 PE have ions at m/z 834.5 and 834.4, respectively.

Figure 3 shows the PC molecular species that are observed in the positive ion mode. NLscanning of 59.1 amu (loss of trimethylamine) and 183.1 amu (loss of phosphocholine),respectively, confirm that these species in the TIC are PC species. The lack of an appreciablechange in the NL mass spectra, plus and minus HCI treatment, when scanning at 59.1 and 183.1amu revealed that the total retina PC pool is devoid of plasmalogens. The presence of prominentions at m/z 756.4, 782.3, 810.4, and 856.4, respectively, revealed that the predominant retinal

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PC molecular species were the diacyl species di-16:0 (11.0 mol% of PC), 16:0–18:1 (16.6 mol% of PC), 18:0–18:1 (7.8 mol% of PC), and 18:0–22:6 (19.5 mol% of PC), respectively.

From the above ESI analyses using direct infusion of retinal lipid extracts, it is clear that ratretina phospholipids are highly enriched in DHA, in good agreement with results obtained inprior studies using different approaches (e.g., GLC of derivatized fatty acids prepared fromlipid extracts, after prior separation of phospholipid classes by thin-layer chromatography andpreparation of 1,2-diacylglyeride acetates (Wiegand and Anderson 1982)). However, due tothe presence of isobaric species, particularly isobaric ions arising from 18:0–22:6 PS anddi-22:6 PE, liquid chromatography was employed to first resolve these phospholipid classes,which were then detected using ESI-MS in the negative ion mode (see Fig. 4, panel A). Underthe conditions employed, PE (peak 1) and PS (peak 2) were well resolved from one another;also, the spectra for PE (Fig. 4B, upper panels, denoted “peak 1”) were similar to those obtainedby direct infusion ESI-MS for the PE molecular species observed by precursor ion scanningat m/z 196 (cf. Fig. 2), while the spectra for PS (Fig. 4B, lower panels, denoted “peak 2”) werecomparable to those obtained by direct infusion ESI-MS for the PS molecular species observedby NL scanning at 87 amu (cf. Fig. 1). Under the chromatographic conditions employed, PCand PS molecular species were not resolved from one another, but eluted from the column asan asymmetric doublet (Fig. 4A, peak 2). However, they were distinguished from one anotherby comparative NL scanning, monitoring the acetate adducts at 74 amu (Fig. 4A, bottomprofile), which are the result of the loss of methyl acetate from choline glycerophospholipids.The relative mol % of the three major phospholipid classes of the two-month old control retinas(in comparison to the total mass of these three classes) were: PC (59.7 mol %), PE (27.4 mol%), and PS (12.9 mol %). By way of comparison (see Fliesler and Anderson 1983, for a review),using two-dimensional thin-layer chromatography and lipid phosphorus determination, itpreviously has been reported that PC accounts for ∼45 mol % of the total glycerophospholipidsof rat retina; plasmalogens only account for about 6% of the total PC. Similarly, PE reportedlyaccounts for ∼32 mol % of the total glycerophospholipids of rat retina, with about 28% of thePE being plasmalogens (vide infra), whereas PS comprises ∼10 mol % of the totalglycerophospholipids of adult rat retina. Hence, our values are in reasonably good agreementwith the existing relevant literature.

Comparative Lipidomic Analysis of Phospholipid Molecular Species from AY9944-Treatedand Age-Matched Control Rat Retinas

The molecular species content of whole retina PS, PE and PC phospholipid classes wasexamined as a function of postnatal age (at 1, 2, and 3 months), comparing AY9944-treatedvs. age-matched control (untreated) rats. Figure 5 shows the results obtained for analysis of PSmolecular species. Comparing the control rat retinas to each other as a function of postnatalage, there was a striking increase in the DHA-containing PS molecular species at two months,compared to one and (to a lesser degree) three months. Notably, the predominant retinal PSmolecular species observed in two- and three-month old rats were 18:0–22:6 and di-22:6;between one and two months postnatal, the levels of the former increased by 3.3-fold, whilethose of the latter increased by nearly 13-fold. In contrast, the AY9944-treated rats did notexhibit this age-dependent DHA enrichment in PS molecular species; if anything, the relativemol % contribution of the dominant 18:0–22:6 PS molecular species was relatively constantat all ages examined, while that of the di-22:6 molecular species remained at the same levelthrough three months of age. The major difference, however, was the relatively striking DHAdeficiency of PS molecular species in AY9944-treated rat retinas, compared to age-matchedcontrols, at two and three months. Looking at the 18:0–22:6 species at two and three months,there were ∼3.3-fold and ∼1.8-fold, statistically significant differences between AY9944-treated vs. control retinas, respectively. However, comparing the di-22:6 PS species, there was

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nearly a 17-fold difference at two months and a ∼7-fold difference at three months betweenAY9944-treated and control retinas.

Figure 6A and 6B show the phosphatidylethanolamine and plasmenylethanolamine molecularspecies, respectively, in the control and AY9944 treated rats. The major change in the retinalethanolamine glycerophospholipid profile in the control rats over the first three postnatalmonths was the 2.4-fold increase in the mass of di-22:6 PE between one and two months,remaining high at three months. In contrast, retinal di-22:6 PE levels in AY9944-treated ratsremained at the same levels at two and three months, compared to the first postnatal month.While no difference was observed at one month, there was a greater than three-fold differencein the di-22:6 PE molecular species at two and three months, comparing AY9944-treated retinasto the corresponding age-matched controls. A 48–56% increase in the 18:0–22:6 PE molecularspecies was also observed as the control rats aged between one and three postnatal months (P< 0.01 for comparisons between one month-old rats with either two or three month-old rats);however, no similar changes were observed for this molecular species in retinas from AY9944-treated rats. In fact, comparing AY9944-treated to control rats, there was ca. 21–23% less 18:0–22:6 PE at two and three months. Conversely, the levels of 18:0–22:4 PE were about two-foldgreater in retinas of AY9944-treated rats, compared to controls, at two and three months;however, while this change was statistically significant, this molecular species represents onlya modest percentage of the total PE pool. It also should be noted that, comparing control versusAY9944-treated rats, no such similar changes were observed in the PE plasmalogen pool(prefix “p” in designated molecular species, Fig. 6B), which also contains molecular speciesenriched with DHA. Direct comparisons between the plasmalogen and diacyl molecularspecies of the ethanolamine glycerophospholipids are shown in Figure 6C. At all ages of theuntreated rats, plasmenylethanolamine molecular species represent ∼26–28% of the totalretinal ethanolamine glycerophospholipid pool. In contrast, in AY9944-treated rats theplasmenylethanolamine molecular species represent 29, 29 and 32% of the retinalethanolamine glycerophospholipid pool in one month-old, two-month old and three-month oldrats, respectively. This increased percentage of plasmenylethanolamine in the total pool ispredominantly due to less phosphatidylethanolamine (PE) molecular species present in theAY9944-treated rats compared to the untreated rats (Fig. 6C).

In comparison to differences in the PS and PE molecular species between AY9944-treated andcontrol rat retinas, the differences in PC molecular species were considerably more complex(see Fig. 7A and 7B). The three most prevalent molecular species of PC in rat retina are 16:0–18:1, 18:0–22:6, and di-16:0. For the 16:0–18:1 molecular species, the levels were significantlylower in AY9944-treated rats than in controls at both one and two months age (Fig 7A). Also,at all three ages examined, the disaturated PC molecular species (di-16:0 and 16:0–18:0) weresignificantly less in AY9944-treated rats, compared to age-matched controls. However, relativeto controls, the difference in di-18:0 PC molecular species was statistically significant inAY9944-dependent only at two months,. It also should be noted that, for these saturated PCmolecular species, there was no age-dependent increase in the control rat retinas. However, incomparison to the content in one-month old control rats, the content of the retinal PC molecularspecies containing DHA (16:0–22:6, 18:0–22:6, and di-22:6) did increase, with statisticalsignificance (P < 0.005) in two-month old control rats and 18:0–22:6 and di-22:6 PCsignificantly (P < 0.05) increased in three-month old rats (Fig. 7B). In striking contrast, therewere no such increases in these DHA-containing molecular species in the retinas of AY9944-treated rats. This results in much larger age-matched differences in DHA-containing PCmolecular species compared to the differences observed in saturated PC species (e.g., di-16:0PC) in control vs. AY9944-treated rats. In fact, as for PS and, to a lesser extent, for PE, theDHA content of PC molecular species was dramatically less in AY9944-treated rats incomparison to age-matched control rats at both two and three months: for 16:0–22:6, thedifference was ∼2.5-fold and ∼1.9-fold, respectively; for 18:0–22:6, it was ∼2-fold and ∼1.4-

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fold, respectively; and for di-22:6, it was ∼4.4-fold and ∼2.6-fold, respectively. In addition,although 22:6–22:5 PC is a quantitatively minor constituent, there was significantly less of thismolecular species at two and three months (∼3-fold and ∼1.9-fold, respectively) in AY9944-treated rats, compared to controls. Plasmenylcholine molecular species have previously beenshown to represent a very minor percentage of the total retinal choline glycerophospholipidpool and are likely dispersed among multiple molecular species. Plasmenylcholine molecularspecies were not detectable under the direct infusion ESI-MS methods applied in theseanalyses. The plasmenylcholine molecular species likely represent <1% of the total ion currentby ESI-MS detection, which limits their detection among many other ion species. In priorreports (reviewed in Fliesler and Anderson 1983), PC plasmalogen detection and analysis wasfacilitated by collapsing the species into one or two peaks (detection by GC with flameionization detection) upon converting the plasmalogen vinyl ethers into 16:0 and 18:0dimethylacetal methanolysis products.

Analysis of Serum and Liver Fatty Acid Composition in AY9944-Treated and Control RatsThe results of the analysis of the total fatty acid composition of serum samples from AY9944-treated vs. control rats as a function of postnatal age are shown in Figure 8. Values for eachacyl species are expressed as relative mole percent of the total. The predominant three fattyacids in serum of both treated and control rats were 16:0, 18:2n6, and 20:4n6. By comparison,18:3n3 and 22:6n3 are relatively minor species, on average accounting for only about 0.3 mol% and 2.3 mol%, respectively, of the total fatty acids in serum (see insets in each panel, Fig.8). The data clearly indicate that AY9944 treatment does not cause a generalized n3 fatty aciddeficiency in rats under the conditions employed. In fact, if anything, the levels of these fattyacids were slightly elevated in sera from the treated animals, relative to controls. At all threetime points, the levels of arachidonic acid (20:4n6) in the treated animals were about half thoseof age-matched controls (P < 0.001). At one and two months of treatment (but not at threemonths), there was a partially compensatory increase in the levels of 18:2n6 and 18:1 in treatedanimals, compared to controls (P < 0.001).

The findings for serum were confirmed and extended by a similar analysis of liver fatty acidcomposition as a function of AY9944 treatment (Fig. 9). In this case, we examined tissues onlyfrom the three-month time point, with the rationale that if AY9944 treatment caused a deficitin DHA or n3 fatty acids in general, it would be apparent in animals treated chronically for thelongest period of time. Also, since the liver is the biogenic source of the DHA that is taken upby and incorporated into the retina (Scott and Bazan 1989), it would be the most likely placeto observe such a deficit. However, as shown in Fig. 9, AY9944 treatment caused no loss ofDHA in the livers of AY9944-treated rats, relative to age-matched controls on the same diet,even after three months of treatment. In fact, the livers of treated animals contained 1.34-foldmore DHA than control livers (P = 0.0285, N=4). The levels of the dominant fatty acyl species,20:4n6, in control livers were only modestly elevated (by 14.6%, P = 0.0087, N=4) relative tothose of AY9944-treated rats. There were no statistically significant changes in the four othermajor fatty acid species (16:0, 18:0, 18:1, and 18:2n6) as a function of AY9944 treatment,which collectively represent about 68% of the total liver fatty acid content.

DISCUSSIONIn the present study, we have demonstrated striking alterations in the phospholipid molecularspecies profile of the retina in rats treated with AY9944, compared to age- and sex-matchedcontrol rats, particularly with respect to the steady-state levels of DHA. The predominantcholine glycerophospholipid molecular species in control retinas were 16:0–22:6 and 18:0–22:6; the levels of these molecular species were lower by ∼30–50% in rats treated for 2–3months with AY9944 in comparison to age-matched control rats. The dominant DHA-

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containing retinal PE molecular species in both control and treated rats were 18:0–22:6 anddi-22:6. In comparison to controls, AY9944 treatment resulted either in no difference (at onemonth), a 22 and 73% differrence (by two months), or a 21% and 69% difference (by threemonths) of these ethanolamine glycerophospholipid molecular species, respectively. Thepredominant retinal PS molecular species were 18:0–22:6 and di-22:6; AY9944 treatmentresulted in >90% less di-22:6 PS, relative to controls. These findings demonstrate that theretinal lipidome is globally altered in the AY9944-treated rat, relative to control rats, over andabove the changes in sterol composition (elevated 7DHC and reduced Chol levels) due to theprimary inhibition of cholesterol biosynthesis caused by AY9944. To the extent that theAY9944-treated rat represents as suitable model of Smith-Lemli-Opitz syndrome, these resultssuggest that additional metabolic compromise beyond the primary enzymatic defect in thecholesterol pathway may be involved in the pathobiology of the human disease.

It should be noted that AY9944 is presumed to be a selective inhibitor of 3β-hydroxysterol-Δ7-reductase (DHCR7; EC 1.3.1.21) (Dvornik et al. 1963; Givner and Dvornik 1965), and hasno reported effects on any enzyme involved in fatty acid or phospholipid biosynthesis. This isconsistent with our finding that the n3 fatty acid content (especially DHA) of plasma and liverwas not reduced in rats treated systemically with AY9944. Also, depletion of retinal DHAlevels was achieved over a relatively brief time frame (2–3 months), even though the dietcontained adequate levels of 18:3n3 (ca. 6 mol% of total fatty acids, as determined by GLCanalysis; data not shown) and both AY9944-treated and control rats were fed the same diet.These findings are all the more remarkable when one considers the well-established fact thatthe vertebrate retina, particularly the outer segment membranes of retinal rod photoreceptorcells, and in striking contrast to other bodily tissues, strongly resists depletion of DHA andother n3 fatty acids when challenged with an n3-deficient diet (Futterman et al. 1971; Andersonand Maude 1972; Tinoco et al. 1977; Wiegand et al. 1991; Anderson et al. 1992; Bush et al.1994; Tinoco et al. 1978). This is thought to be a consequence of an extremely active andefficient recycling mechanism, involving both the retinal pigment epithelium and the neuralretina (Bazan et al. 1992; Bazan et al. 1994; Scott and Bazan 1989).

The retina requires high levels of DHA for optimal function. Reduction of DHA in the ROSof rats (Benolken et al. 1973; Wheeler et al. 1975) and monkeys (Neuringer et al. 1984), bydietary restriction of n3 precursors, leads to reduced amplitudes and other changes in theelectroretinogram. More recent studies have shown that ROS membranes with low DHA levelshave a slower visual response to light (Niu et al. 2004). At the molecular level, this is seen asa slower transition of meta-rhodopsin-I to meta-rhodopsin-II, an important step in the activationof the G-protein, transducin, and subsequent initiation of the visual signal. Studies in term andpre-term human infants have established that the development of visual acuity (Uauy et al.1990; Uauy et al. 1992; Birch et al. 1998) is dependant on a maternal supply of n3 fatty acids.Also, humans and dogs with inherited retinal degenerations have reduced plasma levels of n3PUFAs (Anderson et al. 1987; Anderson et al. 1991). In addition, the retinas of dogs [Aguirreet al. 1997], rats (Anderson et al. 2002), and mice (Anderson et al. 2001; Anderson, Maudeand Bok 2001) with inherited retinal degenerations have significant reductions of DHA in theirROS membranes. These results are of further interest because the loss of DHA is notaccompanied by an increase in 22:5n6, which typically occurs in n3 fatty acid deficiency(Anderson and Maude 1972; Galli et al. 1972; Wiegand et al. 1995). The reason for thisreduction in DHA, even in the presence of adequate levels of dietary n3 fatty acids (which wealso observed in the current study) is unclear. The only other example of a reduction of DHAin the retina without a compensatory replacement by 22:5n6 occurs in albino rats (Penn andAnderson 1987; Penn and Anderson 1992) and mice (Kaldi et al. 2003) raised in bright cycliclight. The reasons for the reduction of DHA in these various animal models are not known.However, it is clear that retinas destined for degeneration, whether by heredity or by light-induced stress, respond by reducing the levels of DHA in their retinas, particularly in

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photoreceptor cell membranes. This is consistent with what we observed in this animal modelof SLOS. These changes are all the more intriguing because they reflect metabolic eventsoccurring specifically in the retina, rather than those occurring in other tissues.

The mechanism by which this extraordinary remodeling of the retinal lipidome is achieved inSLOS rats likely involves multiple aspects of lipid transport and homeostasis, both systemicas well as retina-specific. First, it is possible that differential uptake of essential fatty acids mayoccur in the gut as a consequence of alterations in the composition and content of bile acids(required for solvation of lipids), due to reduced and abnormal sterol biosynthesis caused byAY9944. While not examined specifically in AY9944-treated rats, it has been reported thathuman SLOS patients have altered urinary bile acid composition, both with respect to decreasedlevels of normal bile acids and appearance of abnormal bile acids (Natowicz and Evans1994). This would be expected, since cholesterol is an obligatory precursor of bile acids.However, this finding is somewhat controversial, since a more recent report by Steiner et al.(2000) has shown that while total sterol synthesis was reduced (by about 40%) in their cohortof SLOS patients, bile acid synthesis was not substantially different from control levels, andboth normal primary and secondary bile acids were detected. Second, it is possible that thecontent of lipoprotein particles carrying DHA-enriched lipid constituents, which are assembledin and secreted by the liver, is different (i.e., DHA-deficient) in AY9944-treated vs. controlrats. This is an important consideration, since the retina derives its DHA from the liver, notfrom local de novo synthesis (Scott and Bazan 1989). However, the results of our detailed fattyacid analysis of serum and liver rule out this possibility, since serum DHA levels wereequivalent to (at one and two months) or even slightly higher (at three months) in AY9944-treated animals, and liver DHA levels were slightly elevated at three months, compared tocontrol values (see Fig. 8 and Fig. 9). In addition to this prima facia lack of reduced DHAlevels in serum and liver, systemic n3 fatty acid deficiency in AY9944-treated rats is also ruledout by the fact that there was no observed elevation of 22:5n6 in these tissues, which typicallyoccurs in n3 fatty acid deficiency (see above). If fatty acid absorption in the gut (the firstpossibility mentioned above) was a significant factor, one would expect the fatty acidcomposition of serum and liver to be affected as well. However, given the fact that no DHAor more generalized n3 fatty acid deficiency was apparent in either serum or liver as a functionof AY9944 treatment, the first possibility also tends to be ruled out. This leads to a third, andmost likely, possibility: disruption in the uptake of blood-borne, DHA-containing lipoproteinsby the retina, which also involves the retinal pigment epithelium at the choroidal interface, andsubsequent intraretinal redistribution of the lipid constituents among the various retinal celltypes and metabolic compartments, may be altered in AY9944-treated rats, compared tocontrols. This latter process is complex, involving multiple carriers (both intracellular andextracellular), receptors, and enzymes (Tserentsoodol et al. 2006). Fourthly, there may beadaptive changes in de novo fatty acid synthesis in photoreceptors and other retinal cell typesas a consequence of AY9944 treatment (see below). Resolution of these various possibilitiesis beyond the scope of the present study, but is the subject of active ongoing investigations inour laboratories.

Although DHA-containing phospholipid molecular species were the most profoundly alteredlipids detected in this study, the levels of other fatty acids also were affected by AY9944treatment. To some extent, this could reflect changes in de novo synthesis occurring within theretina per se. Although we have not yet performed metabolic labeling experiments to examinethis further, we have obtained preliminary evidence, using microarray analysis, that indicatesthe expression levels of several genes involved in fatty acid and phospholipid biosynthesis aresignificantly altered in retinas of AY9944-treated rats, compared to controls, particularly aftertwo months of treatment (Siddiqui et al. 2007). These include, but are not limited to, fatty acidsynthase, stearoyl-CoA desaturase, acyl-CoA thioesterase, acyl-CoA synthetase, and CDP-diacylglycerol synthase. Since AY9944 has no known direct effect on any of these enzymes,

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it is conceivable that cross-talk between the cholesterol biosynthetic pathway and the fatty acidand phospholipid biosynthetic pathways, mediated via sterol response elements and theSREBP/SCAP system (reviewed in Shimano 2001; Rawson 2003; McPherson and Gauthier2004), may explain, at least in part, these observations.

It should be appreciated that by one month of AY9944 treatment, there is no retinaldegeneration and no electrophysiological dysfunction of the retina; yet, at the same time, retinalsterol composition is grossly deranged, with the 7DHC/Chol mole ratio being nearly 4:1(Fliesler et al. 1999). In the present study, we found that alterations to retinal phospholipidmolecular species after one month of AY9944 treatment were minimal. However, by threemonths of AY9944 treatment, substantial retinal degeneration and dysfunction accompanieseven greater derangement of sterol metabolism and composition (Fliesler et al. 2004). Here,we found that retinal phospholipid molecular species composition is dramatically altered within2–3 months of AY9944 treatment, compared to age-matched controls. Hence, phospholipidcomposition seems to correlate more closely with retina histological and electrophysiologicalintegrity than does sterol composition in this animal model. However, it should not be inferredfrom these findings that reduced levels of DHA somehow caused the observed retinaldegeneration. [Similarly, in the cases of hereditary retinal degeneration in animals, asmentioned above, there is no evidence that reduced levels of DHA cause the retinaldegeneration.] A correlation between n3 fatty acid content and retinal development andfunction has been established for many years, both for rats (Benolken et al. 1973; Wheeler etal. 1975) as well as humans and non-human primates (Neuringer et al. 1984; Uauy et al.1990; Uauy et al. 1992; Birch et al. 1998). However, given the fact that we found no evidencefor a systemic n3 fatty acid deficiency in this SLOS rat model, it is highly unlikely that feedinga high n3-containing (e.g., 18:3n3- or 22:6n3-rich) diet in addition to cholesterolsupplementation, as opposed to a high-cholesterol diet alone, would provide any additionalbenefit with regard to improving retinal structure or function in the SLOS rat model. Indeed,even when AY9944-treated rats are fed a high-cholesterol diet for two months, which tends tonearly normalize their serum and retina sterol compositions, there is no protection againstretinal degeneration (Fliesler et al. 2007). Furthermore, in a study by Martin et al. (2004), itwas found that although supplementation with a diet enriched in n3 fatty acids was able to alterand partially restore the normal steady-stated fatty acid profile of the retina (and, morespecifically, of rod photoreceptor outer segment membranes), such dietary manipulation wasneither able to protect against photoreceptor cell death nor alter the course of hereditary retinaldegeneration in two different rat models carrying rhodopsin mutations linked to human retinaldisease. In fact, the retina is known to conserve DHA under conditions of dietary n3 fatty aciddeficiency (Anderson and Maude 1972; Wiegand et al. 1991), and even the small amounts ofDHA present in standard rat chow are normally sufficient to support the high levels of DHAtypically found in retinal photoreceptor membranes (Benolken et al. 1973). Taken together,these findings would suggest that the mechanism underlying the profound decrease in retinalDHA levels in the AY9944-treated rat model of SLOS is fundamentally different from thatwhich underlies retinal DHA decline under conditions of systemic n3 fatty acid deficiency(e.g., as induced by dietary manipulation).

The findings obtained with this AY9944-treated rat model would predict that the retinas ofSLOS patients may have altered fatty acid composition, particularly abnormally low DHAlevels, compared to unaffected, age-matched normal individuals. Unfortunately, there are noreports in the literature, at present, to either confirm or negate this prediction, and obtainingSLOS as well as normal human retinal tissue specimens is extraordinarily difficult. However,we currently are pursuing a study of the serum fatty acid profiles of SLOS patients, comparingthem with normal controls, to ascertain if there is any correlation between disease severity andserum fatty acid profile. Our findings with the AY9944 rat model would predict a lack of suchcorrelation.

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Abbreviations7DHC, 7-dehydrochosterolamu, atomic mass unitsBHT, butylated hydroxytolueneChol, cholesterolDHA, docosahexaenoic acidDTPA, diethylenetriamine pentaacetic acidESI-MS, electrospray ionization mass spectrometryFAMES, fatty acid methyl estersGLC, gas-liquid chromatographyLC/MS, liquid chromatography/mass spectrometry (LC/MS)NL, neutral lossPC, phosphatidylcholinePE, phosphatidylethanolaminePS, phosphatidylserinePUFAs, polyunsaturated fatty acidsSLOS, Smith-Lemli-Opitz syndromeTIC, total ion current

ACKNOWLEDGMENTSThis study was supported by U.S.P.H.S. (NIH) grants EY007361 (SJF), HL74214 (DAF), RR019232 (DAF), EY00871(REA), EY04149 (REA), EY12190 (REA), RR17703 (REA), Foundation Fighting Blindness (REA), and unrestricteddepartmental grants from Research to Prevent Blindness (SJF and REA). SJF is the recipient of a Research to PreventBlindness Senior Scientific Investigator Award.

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FIGURE 1.ESI-MS analysis (in the negative ion mode) of phospholipids from a two-month old normalrat retina. Following Bligh-Dyer extraction, the organic extract was subjected to ESI-MS asdescribed in Experimental Procedures. For all spectra, samples were directly infused into theESI source at a flow rate of 3 µl/min. The top spectrum was acquired in the negative-ion modedirectly from a lipid extract that was diluted to less than 20 pmol of total lipids per microliter(4:1, v/v, methanol/chloroform). The same samples were also subjected to tandem massspectrometry with neutral loss (NL) scanning for 87 (for PS molecular species) or precursorion scanning for either 153, 283.2, 303.3 or 327.3 amu. All mass spectral traces are displayedafter normalization to the base peak in each individual spectrum.

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FIGURE 2.ESI-MS analyses (negative ion mode) of ethanolamine glycerophospholipids from a two-month old normal rat retina. Conditions as in legend, Fig. 1, except with the addition ofapproximately 10 pmol NaOH per microliter to the lipid extract prior to analysis. The samesamples were also subjected to tandem mass spectrometry with precursor ion scanning foreither 196 (for PE molecular species), 283.2, 303.3 or 327.3 amu. Spectra were also acquiredfrom lipid extract that was first dried and treated for 45 sec with HCI vapors prior to preparationfor mass spectrometry (HCI treated). All mass spectral traces are displayed after normalizationto the base peak in each individual spectrum.

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FIGURE 3.ESI-MS analyses (positive ion mode) of choline glycerophospholipids from a two-month oldnormal rat retina. Conditions as describe in legend, Fig. 1, except using the positive ion mode,and the lipid extract was treated with approximately 10 pmol NaOH per microliter prior toanalysis. The same samples were also subjected to tandem mass spectrometry with NL scanningfor either 59.1 or 183.1 amu (both for PC molecular species). Spectra were also acquired fromlipid extract that was first dried and treated for 45 sec with HCI vapors prior to preparation formass spectrometry (HCI treated). All mass spectral traces are displayed after normalization tothe base peak in each individual spectrum.

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FIGURE 4.LC/MS analyses of phospholipids (in the negative ion mode) extracted from a 2-mo old normalrat retina. Following Bligh-Dyer extraction, the organic extract was subjected to normal-phaseliquid chromatography and ESI-MS detection as described in Experimental Procedures. PanelA: Total ion currents for the chromatogram, for the mass range from m/z 600–950 (top) andthat using precursor ion scanning for 196 amu or NL scanning for either 87 or 74 amu asindicated. Panel B: Corresponding mass spectra of the regions labeled as peaks 1 and 2(chromatogram, Panel A). For each of these peaks, the mass spectrum of the negative ions areshown in the upper portion, which the mass spectrum using either the precursor ion or NLscanning technique are depicted in the lower portion of each panel.

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FIGURE 5.Serine glycerophospholipid (PS) molecular species composition of retinas from normal(hatched bars) and AY9944-treated (filled bars) rats as a function of postnatal age [1 month(black bars), 2 months (blue bars), and 3 months (red bars)]. Lipid extracts were subjected toESI-MS with direct infusion, essentially per the conditions given in legend, Fig. 1. Individualmolecular species were quantified by comparisons to the internal standard as described inExperimental Procedures. Due to overlap with di-22:6 phosphatidylethanolamine, 18:0–22:6PS was quantified using NL scanning of 87 amu, as indicated. Asterisk (*) indicates statisticalsignificance (P < 0.05, Student’s t-test; N=3) for comparison of a given molecular speciesbetween AY9944-treated vs. control rats at the same age.

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FIGURE 6.Ethanolamine glycerophospholipid molecular species composition of retinas from normal(hatched bars) and AY9944-treated (filled bars) rats as a function of postnatal age [1 month(black bars), 2 months (blue bars), and 3 months (red bars)]. Lipid extracts were treated withaddition of approximately 10 pmol NaOH per microliter and then subjected to ESI-MS withdirect infusion, essentially per the conditions given in legend, Fig. 1. Individualphosphatidylethanolamine (A) and plasmenylethanolamine (B) molecular species werequantified by comparisons to the internal standard as described in Experimental Procedures.Due to overlap with 18:0–22:6 PS, di-22:6 phosphatidylethanolamine was quantified usingprecursor ion scanning of 196 amu as indicated. The sum of total phosphatidylethanolamine

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versus total plasmenylethanolamine molecular species is shown in panel C. Statisticaldifferences at *P < 0.05 and **P < 0.005 (Student’s t-test; N=3) are indicated for comparisonof a given molecular species between AY9944-treated and control rats at the same age.

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FIGURE 7.Choline glycerophospholipid (PC) molecular species composition of retinas from normal(hatched bars) and AY9944-treated (filled bars) rats as a function of postnatal age [1 month(black bars), 2 months (blue bars), and 3 months (red bars)]. Lipid extracts were treated withaddition of approximately 10 pmol NaOH per microliter and then subjected to ESI-MS withdirect infusion, essentially per the conditions given in legend, Fig. 1, except in the positive ionmode. Individual molecular species including those without PUFAs (A) and those containingPUFAs (B) were quantified by comparisons to the internal standard as described inExperimental Procedures. Statistical differences at *P < 0.05 and **P < 0.005 (Student’s t-

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test; N=3) are indicated for comparison of a given molecular species between AY9944-treatedand control rats at the same age.

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FIGURE 8.Serum fatty acid composition of control (open bars) and AY9944-treated rats (filled bars) asa function of postnatal age. (A) 1 month; (B) 2 months; (C) 3 months. Total lipids wereextracted and the corresponding fatty acid methyl esters (FAMEs) were prepared therefromand analyzed by GLC (see Experimental Procedures for details). Values are expressed asrelative mol% (mean ± S.D., N=4). Statistical significance was evaluated using multivariantANOVA with a post-hoc Scheffe test (*P < 0.05; **P < 0.01; ***P < 0.001).

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FIGURE 9.Liver fatty acid composition of three-month old control (open bars) and AY9944-treated rats(filled bars). The treated rats received AY9944 throughout their entire lifetime. Total lipidswere extracted and the corresponding fatty acid methyl esters (FAMEs) were preparedtherefrom and analyzed by GLC (see Experimental Procedures for details). Values areexpressed as relative mol% (mean ± S.D., N=4). Statistical significance was evaluated usinga two-tailed Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001).

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TABLE 1Sterol composition and content of serum from AY9944-treated and control ratsAliquots (50 µl each) of serum were saponified in methanolic KOH and the nonsaponifiable lipids were extracted withpetroleum ether, evaporated to dryness, redissolved in methanol, and analyzed by reverse-phase HPLC. Valuesrepresent the mean ± S.D., with number of biologically independent samples (N) given in parentheses, corrected forrecovery efficiency using an internal standard of [3H]cholesterol. 7DHC, 7-dehydrocholesterol; Chol, cholesterol.

Treatment Group Age (mo) 7DHC/Chol (mole ratio) Total Sterols (mg/dl) Total Sterols(% of Control)

Control 1 (N=7) 0 100.5 ± 10.7 100

+AY9944 1 (N=19) 2.0 ± 0.8 16.5 ± 6.2 16.4

Control 3 (N=12) 0 95.5 ± 17.5 100

+AY9944 3 (N=16) 4.6 ± 1.0 20.8 ± 5.6 21.8


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Lipidomic analysis of the retina in a rat model of Smith–Lemli–Opitz syndrome: alterations in docosahexaenoic acid content of phospholipid molecular species