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Keywords:

  • AY9944;
  • docosahexaenoic acid;
  • fatty acid;
  • lipidomic analysis;
  • retina

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Smith–Lemli–Opitz syndrome (SLOS) is a complex hereditary disease caused by an enzymatic defect in the last step of cholesterol biosynthesis. Progressive retinal degeneration occurs in an AY9944-induced rat model of SLOS, with biochemical and electroretinographic hallmarks comparable with the human disease. We evaluated alterations in the non-sterol lipid components of the retina in this model, compared with age-matched controls, using lipidomic analysis. The levels of 16:0–22:6 and 18:0–22:6 phosphatidylcholine molecular species in retinas were less by > 50% and > 33%, respectively, in rats treated for either 2 or 3 months with AY9944. Relative to controls, AY9944 treatment resulted in > 60% less di-22:6 and > 15% less 18:0–22:6 phosphatidylethanolamine molecular species. The predominant phosphatidylserine (PS) molecular species in control retinas were 18:0–22:6 and di-22:6; notably, AY9944 treatment resulted in > 80% less di-22:6 PS, 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 the most profound changes being less phosphatidylcholine, phosphatidylethanolamine, and PS molecular species containing docosahexaenoic acid (22:6). These findings suggest that SLOS may involve additional metabolic compromise beyond the primary enzymatic defect in the cholesterol pathway.

Abbreviations used
7DHC

7-dehydrocholesterol

amu

atomic mass units

Chol

cholesterol

DHA

docosahexaenoic acid

ESI-MS

electrospray ionization-mass spectrometry

FAME

fatty acid methyl ester

LC

liquid chromatography

NL

neutral loss

PBS

phosphate-buffered saline

PC

phosphatidylcholine

PE

phosphatidylethanolamine

PS

phosphatidylserine

ROS

rod outer segments

SLOS

Smith–Lemli–Opitz syndrome

SREBP/SCAP

sterol regulatory element-binding protein/SREBP cleavage-activating protein

TIC

total ion current

Smith–Lemli–Opitz syndrome (SLOS) is an autosomal recessive, multiple congenital anomalies disorder caused by a defect in the last enzymatic step in the cholesterol (Chol) biosynthetic pathway (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 a specific biochemical signature: blood and other tissues contain abnormal (typically grossly elevated) levels of 7-dehydrocholesterol (7DHC) and markedly reduced levels of Chol, relative to the levels found in normal controls (Irons et al. 1993; Tint et al. 1994). This is due to mutations in the DHCR7 gene, which encodes the enzyme 3β-hydroxysterol-Δ7-reductase (EC 1.3.1.21) (Fitzky et al. 1998; Waterham et al. 1998; Correa-Cerro and Porter 2005; Yu and Patel 2005), thereby altering the efficiency of conversion of 7DHC to Chol. The nervous system, in particular, is profoundly affected in this disease, as manifested by cognitive function deficits (e.g. mental retardation and 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 animal model of this human disease has been developed by treating rats with AY9944, a selective pharmacological inhibitor of the same enzyme that is defective in SLOS (Dvornik et al. 1963; Givner and Dvornik 1965; Kolf-Klauw et al. 1996; Wolf et al. 1996). Using a modification of the originally developed model that allows long-term postnatal survival, a progressive retinal degeneration affecting both rod and cone photoreceptors have been described (Fliesler et al. 2004). Whereas the normal rat retina contains virtually no 7DHC, the mole ratio of 7DHC/Chol in retinas of 1-month-old rats treated with AY9944 as 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 high-Chol diet to these rats from weaning through three postnatal months provides a marked improvement primarily in cone photoreceptor function, and also significantly improves (toward normal) the sterol profile of 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 examined the 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 report herein that the fatty acid composition of the major retinal phospholipid molecular species is dramatically altered in the SLOS rat model, relative to that of controls. Remarkably, the relative docosahexaenoic acid (DHA; 22:6n3) content of these phospholipids was significantly reduced within a 3-month time course, to an extent comparable with or greater than that achieved heretofore by raising rats on a n3 fatty acid-deficient diet (Futterman et al. 1971; Anderson and 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 no evidence of generalized, systemic DHA or n3 fatty acid deficiency. We discuss these findings within the context of the role of lipids in supporting normal visual function, and also with regard to the pathobiology of SLOS. To the extent that the AY9944-induced rat model faithfully mimics the human disease, these findings suggest that the metabolic defect in SLOS may involve other pathways beyond the primary defect in the Chol pathway.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

Authentic glycerophospholipid standards were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Fatty acid standards were used as purchased from (Nu-Chek Prep, Inc., Elysian, MN, USA). Sterol standards were obtained from Steraloids, Inc. (Newport, RI, USA); 7DHC was periodically recrystallized from methanol–water and its purity verified by HPLC prior to use. AY9944 [trans-1,4-bis(2-chlorobenzylamino-methyl)cyclohexane dihydrochloride] was custom synthesized, and matched the spectroscopic and physical properties of an authentic sample of AY9944 (kindly provided by Wyeth-Ayerst Laboratories, Monmouth, NJ, USA). All organic solvents were of HPLC grade, and used as purchased from Fisher Scientific (Pittsburgh, PA, USA). Unless otherwise specified, all other reagents were used as purchased from Sigma/Aldrich (St Louis, MO, USA).

Animals

Pregnant Sprague–Dawley rats (6 days sperm-positive) were obtained from Harlan (Indianapolis, IN, USA). Rats (both control and treated) were fed water and a standard rat chow (Purina Mills TestDiet, Richmond, IN, USA), ad lib. The levels of Chol in this chow were below the detectable limit (10 ppm; S. J. Fliesler, data not shown). Rats were treated with AY9944 as previously described (Fliesler et al. 2004, 2007). In brief, pregnant rats implanted with subcutaneous Alzet® osmotic pumps (Model 2ML4; Durect Corporation, Cupertino, CA, USA) containing phosphate-buffered saline (PBS) solution of AY9944 (1.5 mg/mL), so as to deliver the drug at a constant rate (0.37 mg/kg/day, at 2.5 μL/h) from gestational day 7 through the second postnatal week. Control dams received the same food and water ad lib, but were given no other treatment. Pups from AY9944-treated dams were injected subcutaneously three times per week, on alternating days, with AY9944 (30 mg/kg, in PBS), starting at postnatal day 1 and continuing throughout life. Control pups were not treated with any injections, since prior studies (unpublished results; cf. Fliesler et al. 1999) showed no effect of parallel vehicle injections with regard to tissue biochemistry, histology, or retinal function. All procedures involving animals were approved by the Saint Louis University IACUC and conformed to the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and the Association for Research in Vision and Ophthalmology’s Statement for the Use of Animals in Ophthalmic and Visual Research.

Retinal lipid extraction

Whole retinas were harvested from rats in each group (experimental and control) and were immediately frozen in liquid nitrogen in the presence of argon-purged, chilled PBS containing diethylenetriamine pentaacetic acid (0.1 mol/L), butylated hydroxytoluene (0.01 mg/mL), and SnCl2 (0.01 mg/mL). Retinas were subsequently extracted by a modified Bligh–Dyer technique (Ward et al. 1996) in the presence of internal standards. In brief, lipids from one retina were extracted in a Teflon/glass homogenizer using 2 mL of methanol/chloroform (1 : 1, by vol) with phase separation by the addition of 1.5 mL of saline. The methanol/chloroform mixture contained the following internal standards: di-14:0 phosphatidylethanolamine (PE; 23.6 nmol), di-17:0 PE (23.6 nmol), di-20:0 phosphatidylcholine (PC; 11.8 nmol), and di-14:0 phosphatidylserine (PS; 1.8 nmol). Lipids were extracted twice from the retinas and the pooled chloroform layers were washed once with 0.9% (w/v) saline prior to evaporation of solvent under a nitrogen stream and re-suspended in chloroform. Lipid extracts were stored under argon at −85°C in darkness until ready for analysis.

Serum and liver lipid extraction and fatty acid analysis

Whole blood was collected by cardiac puncture from deeply anesthetized animals, and serum was prepared therefrom by centrifugation (5 min at 13 000 g), aliquoted into polypropylene microfuge tubes, flash frozen in liquid nitrogen, and stored at −85°C until ready for analysis. Samples were protected from exposure to light as much as possible throughout processing. After thawing, a mixture containing 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 extracted essentially per the Bligh–Dyer method (Bligh and Dyer 1959), with the minor modifications as described previously by Martin et al. (2005). Lipid extracts were dried under a stream of nitrogen and fatty acids were derivatized to form the corresponding methyl esters (fatty acid methyl esters; FAMEs), prior to analysis by GLC, essentially as described previously (Martin et al. 2005), with the following modifications. In brief, toluene (0.20 mL) plus 1 mL of 2% (by vol) methanolic sulfuric acid were added to each lipid extract, the mixture was sealed in a glass tube under nitrogen atmosphere with teflon-lined caps, vortexed, and heated for 1 h at 100°C. After cooling on ice, 1.2 mL of water was added, and the FAMEs were extracted three times with 2.4 mL of hexane, then dried under nitrogen and dissolved in 20 μL nonane. Fatty acid composition was then determined by injecting 3 μL of each sample onto a DB-225 capillary column (30 m × 0.32 mm I.D.; J&W Scientific, Folsom, CA, USA), using an Agilent 6890N gas chromatograph with model 7683 autosampler (Agilent Technologies, Wilmington, DE, USA), at an inlet temperature of 250°C and a split ratio of 25 : 1. The column temperature was programmed to begin at 160°C, ramped to 220°C at 1.33°C/min, and held at 220°C for 18 min. Hydrogen carrier gas flowed at 1.6 mL/min and the flame ionization detector temperature was set to 270°C. The chromatographic peaks were integrated and processed with ChemStation® software (Agilent Technologies). FAMEs were identified by comparison of their relative retention times with authentic standards and relative mole percentages 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 in the homogenate were pelleted by centrifugation at 1000 g for 10 min and the lipid extract removed. The pellet was washed twice with 1 mL chloroform/methanol (1 : 1, by vol) and the washes were combined with the initial lipid extract. The combined lipid extract was then washed with 0.2 volumes of 1 mmol/L aqueous diethylenetriamine pentaacetic acid followed by Folch theoretical upper phase (chloroform/methanol/water, 3 : 48 : 47, by vol), each time discarding the aqueous upper phase. 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 analysis as described above for serum fatty acid analysis.

Electrospray ionization-mass spectrometry

Extracted retinal lipids were re-suspended in methanol/chloroform (4 : 1, by vol) to a dilution of 20 pmol of lipid per microliter and analyzed by electrospray ionization-mass spectrometry (ESI-MS) in the direct infusion mode at a flow rate of 1–3 μL/min using a Thermo Electron TSQ Quantrum Ultra® instrument (Williams 2000; Albert et al. 2007) (Waltham, MA, USA). Under selected conditions, 10 pmol of NaOH per microliter was added to samples immediately prior to injection (unless indicated otherwise) and samples were run either in the positive or negative ion mode. In the positive ion mode, electrospray needle voltage was 4 kV, and capillary temperature was 280°C. In the negative ion mode, electrospray needle voltage was 3.2 kV, and capillary temperature was 250°C. Tandem MS was performed on selected ions (typical collision energies were ∼28–35 eV) and spectra were averaged over 3–5 min and processed utilizing Xcalibur® (Thermo Electron) software. For choline glycerophospholipids (PC and corresponding plasmalogens), neutral loss (NL) scanning of 59.1 and 183.1 atomic mass units (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 collision energies of 50 eV (for PE) and 35 eV (for fatty acids). Also in the negative ion mode, NL scanning for 87 amu (for PS) and precursor ion scanning for m/z 153 was monitored at collision energies of 25 and 35 eV, respectively. Spectra were averaged over 3–5 min and processed utilizing Xcalibur® software (Thermo Electron). Individual molecular species were quantified by comparing the ion intensity of individual molecular species to that of the appropriate internal standards following corrections for type I and type II 13C isotope effects (Han and Gross 2005). Values are expressed as the mass of each individual molecular species per retina.

Liquid chromatography/mass spectrometry analyses

Retinal lipid extracts were separated on a Thermo Finnigan Surveyor liquid chromatography (LC) equipped with a Hypersil® 150 mm × 1 mm silica column (Thermo) equilibrated with mobile phase A (hexane/isopropanol/1 mol/L ammonium acetate in water, 30/40/2, by vol) at a flow rate of 60 μL/min. Following injection of lipid extracts onto the column, the column was eluted for 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 stationary phase was further eluted for another 25 min with mobile phase A. Lipids eluted from the column were monitored using a TSQ Quantum Ultra triple quadruple mass spectrometer in the negative ion mode (electrospray needle voltage, 4 kV; capillary temperature, 275°C). Lipids were detected as negative ions as well as through MS/MS techniques, including precursor ion scanning for m/z 196 as well as NL scanning for both 87 amu (for PS) and 74 amu (for PC: loss of methyl acetate from the [M + H3CCOO]).

Sterol analysis

Whole blood was collected from animals at one and three postnatal months, allowed to clot (at 4°C, in darkness), and serum was prepared therefrom by centrifugation (5 min at 13 000 g). Sterol composition was analyzed by reverse-phase HPLC, after saponification and extraction of the non-saponifiable lipids, as previously described (Fliesler et al. 1993, 1999). In brief, each specimen (50 μL total, plus an internal standard of [3H]Chol) was saponified in methanolic KOH, and the non-saponifiable lipids were extracted with petroleum ether, redissolved in methanol, and analyzed by reverse-phase HPLC (detection at 205 nm). Identification of sterols was performed in comparison with authentic standards of 7DHC and Chol; integrated peak areas were analyzed with respect to empirically determined response factors for each sterol, with calculated masses corrected for recovery efficiency, based upon the recovery of [3H]Chol.

Statistical analyses

For the ESI-MS studies, statistical analysis of the data was performed with respect to the effect of AY9944 treatment versus controls for a given molecular species at the same age (e.g. levels of di-22:6 PS at 2 months postnatal, treated vs. control rats), using a two-tailed Student’s t-test with a maximum cutoff for statistical significance of < 0.05. For serum fatty acid analyses performed by GLC, multivariant anova with post hoc Scheffe tests were used to determine statistical significance, with a maximum cutoff of < 0.05. For the liver fatty acid analyses, statistical significance was evaluated using a two-tailed Student’s t-test, with a maximum cutoff of < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Global disruption of sterol biosynthesis by AY9944

To verify that AY9944-treated animals exhibited the expected alteration of sterol metabolism consistent with the biochemical hallmarks of SLOS (i.e. accumulation of 7DHC and reduction in levels of Chol), serum samples collected at one and three postnatal months were analyzed quantitatively 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 of treatment time: at 1 month, the ratio was 2.0 ± 0.8, while at 3 months, the ratio was 4.6 ± 1.0. Serum from control rats had undetectable levels of 7DHC. Also, in agreement with the fact that AY9944 is a potent hypocholesterolemic drug (Dvornik et al. 1963; Givner and Dvornik 1965; Kolf-Klauw et al. 1996; Wolf et al. 1996), the total sterol content of serum at 1 and 3 months in AY9944-treated rats was 16.5 ± 6.2 and 20.8 ± 5.6 mg/dL, respectively, compared with 100.5 ± 10.7 and 95.5 ± 17.5 mg/dL in serum samples from control rats at one and three postnatal months, respectively. On average, AY9944 treatment caused a ca. fivefold reduction in total serum sterol levels, compared with controls, over the specified treatment period. Hence, under the conditions employed, AY9944-treated animals faithfully mimicked the signature biochemical hallmarks of SLOS, in good agreement with prior studies (Dvornik et al. 1963; Givner and Dvornik 1965; Kolf-Klauw et al. 1996; Wolf et al. 1996; Fliesler et al. 1999, 2004).

Table 1.   Sterol composition and content of serum from AY9944-treated and control rats
Treatment groupAge (months) 7DHC/Chol (mole ratio)Total sterols (mg/dL)Total sterols (% of control)
  1. Aliquots (50 μL each) of serum were saponified in methanolic KOH and the non-saponifiable lipids were extracted with petroleum ether, evaporated to dryness, redissolved in methanol, and analyzed by reverse-phase HPLC. Values represent the mean ± SD, with number of biologically independent samples (n) given in parentheses, corrected for recovery efficiency using an internal standard of [3H]cholesterol. 7DHC, 7-dehydrocholesterol; Chol, cholesterol.

Control1 (n = 7)0100.5 ± 10.7100
+AY99441 (n = 19)2.0 ± 0.816.5 ± 6.216.4
Control3 (n = 12)095.5 ± 17.5100
+AY99443 (n = 16)4.6 ± 1.020.8 ± 5.621.8

Shotgun lipidomic analysis of phospholipids from normal adult rat retina

As a prelude to a detailed comparative analysis of the retinal phospholipid composition of AY9944-treated versus age-matched control rats, we used a shotgun lipidomics approach (Han and Gross 2005) to examine phospholipid composition in the adult, normal rat retina. Figure 1 shows the negative ion spectra of retinal phospholipids from a 2-month-old normal Sprague–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 mass spectrum of the total ion current (TIC) revealed that the predominant molecular species observed under these conditions are those arising from PS and phosphatidylinositol molecular species. The PS molecular species were specifically detected by NL scanning of 87 amu (Fig. 1). Precursor ion scanning of m/z 153 showed all species containing a glycerol phosphate moiety, which includes not only PS species, but also phosphatidylinositol molecular species. There were three predominant PS molecular species, containing the following diacyl pairs (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, 834.5, and 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 spectra in comparison with the weakly anionic PE molecular species. Because we were interested primarily in the three major glycerophospholipid classes (PC, PE, and PS), we did not include an internal phosphatidylinositol standard in the samples. Hence, one cannot reliably quantify the relative mole percentage of phosphatidylinositols based upon the peak intensities shown in Fig. 1. Prior studies (reviewed in Fliesler and Anderson 1983) indicate that phosphatidylinositol accounts for only ca. 4% of the total phospholipids in rat retina. Additionally, precursor ion scanning of m/z 283.2, 303.3, and 327.4 (Fig. 1) was used to identify the individual esterified fatty acids in these phospholipids, which correspond to stearic acid (18:0), arachidonic acid (20:4), and DHA (22:6), respectively.

image

Figure 1.  ESI-MS analysis (in the negative ion mode) of phospholipids from a 2-month-old normal rat retina. Following Bligh–Dyer extraction, the organic extract was subjected to ESI-MS as described in Experimental procedures. For all spectra, samples were directly infused into the ESI source at a flow rate of 3 μL/min. The top spectrum was acquired in the negative-ion mode directly 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 mass spectrometry with neutral loss (NL) scanning for 87 (for PS molecular species) or precursor ion scanning for either 153, 283.2, 303.3, or 327.3 amu. All mass spectral traces are displayed after normalization to the base peak in each individual spectrum.

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

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

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Figure 3 shows the PC molecular species that are observed in the positive ion mode. NL scanning 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 appreciable change in the NL mass spectra, plus and minus HCl treatment, when scanning at 59.1 and 183.1 amu revealed that the total retina PC pool is devoid of plasmalogens. The presence of prominent ions at m/z 756.4, 782.3, 810.4, and 856.4, respectively, revealed that the predominant retinal PC molecular species were the diacyl species di-16:0 (11 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.

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Figure 3.  ESI-MS analyses (positive ion mode) of choline glycerophospholipids from a 2-month-old normal rat retina. Conditions as describe in legend, Fig. 1, except using the positive ion mode, and the lipid extract was treated with ∼10 pmol NaOH per microliter prior to analysis. The same samples were also subjected to tandem mass spectrometry with NL scanning for either 59.1 or 183.1 amu (both for PC molecular species). Spectra were also acquired from lipid extract that was first dried and treated for 45 s with HCl vapors prior to preparation for mass spectrometry (HCl treated). All mass spectral traces are displayed after normalization to the base peak in each individual spectrum.

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From the above ESI analyses using direct infusion of retinal lipid extracts, it is clear that rat retina phospholipids are highly enriched in DHA, in good agreement with results obtained in prior studies using different approaches (e.g. GLC of derivatized fatty acids prepared from lipid extracts, after prior separation of phospholipid classes by TLC and preparation of 1,2-diacylglyceride acetates (Wiegand and Anderson 1982). However, because of the presence of isobaric species, particularly isobaric ions arising from 18:0 to 22:6 PS and di-22:6 PE, LC was employed to first resolve these phospholipid classes, which were then detected using ESI-MS in the negative ion mode (see Fig. 4a). Under the 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 obtained by direct infusion ESI-MS for the PE molecular species observed by precursor ion scanning at m/z 196 (cf.Fig. 2), while the spectra for PS (Fig. 4b, lower panels, denoted ‘Peak 2’) were comparable with those obtained by direct infusion ESI-MS for the PS molecular species observed by NL scanning at 87 amu (cf.Fig. 1). Under the chromatographic conditions employed, PC and PS molecular species were not resolved from one another, but eluted from the column as an asymmetric doublet (Fig. 4a, peak 2). However, they were distinguished from one another by comparative NL scanning, monitoring the acetate adducts at 74 amu (Fig. 4a, bottom profile), which are the result of the loss of methyl acetate from choline glycerophospholipids. The relative mol% of the three major phospholipid classes of the 2-month-old control retinas (in comparison with 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 TLC and lipid phosphorus determination, it previously has been reported that PC accounts for ∼45 mol% of the total glycerophospholipids of rat retina; plasmalogens only account for about 6% of the total PC. Similarly, PE reportedly accounts for ∼32 mol% of the total glycerophospholipids of rat retina, with about 28% of the PE being plasmalogens (vide infra), whereas PS comprises ∼10 mol% of the total glycerophospholipids of adult rat retina. Hence, our values are in reasonably good agreement with the existing relevant literature.

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Figure 4.  LC/MS analyses of phospholipids (in the negative ion mode) extracted from a 2-month-old normal rat retina. Following Bligh–Dyer extraction, the organic extract was subjected to normal-phase LC and ESI-MS detection as described in Experimental procedures. (a) Total ion currents for the chromatogram, for the mass range from m/z 600–950 (top) and that using precursor ion scanning for 196 amu or NL scanning for either 87 or 74 amu as indicated. (b) Corresponding mass spectra of the regions labeled as peaks 1 and 2 (chromatogram, a). For each of these peaks, the mass spectrum of the negative ions are shown in the upper portion, which the mass spectrum using either the precursor ion or NL scanning technique are depicted in the lower portion of each panel.

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Comparative lipidomic analysis of phospholipid molecular species from AY9944-treated and age-matched control rat retinas

The molecular species content of whole retina PS, PE, and PC phospholipid classes was examined as a function of postnatal age (at 1, 2, and 3 months), comparing AY9944-treated versus age-matched control (untreated) rats. Figure 5 shows the results obtained for analysis of PS molecular species. Comparing the control rat retinas to each other as a function of postnatal age, there was a striking increase in the DHA-containing PS molecular species at 2 months, compared with 1 and (to a lesser degree) 3 months. Notably, the predominant retinal PS molecular species observed in 2- and 3-month-old rats were 18:0–22:6 and di-22:6; between 1 and 2 months postnatal, the levels of the former increased by 3.3-fold, while those of the latter increased by nearly 13-fold. In contrast, the AY9944-treated rats did not exhibit this age-dependent DHA enrichment in PS molecular species; if anything, the relative mol% contribution of the dominant 18:0–22:6 PS molecular species was relatively constant at all ages examined, while that of the di-22:6 molecular species remained at the same level through 3 months of age. The major difference, however, was the relatively striking DHA deficiency of PS molecular species in AY9944-treated rat retinas, compared with age-matched controls, at 2 and 3 months. Looking at the 18:0–22:6 species at 2 and 3 months, there were ∼3.3-fold and ∼1.8-fold, statistically significant differences between AY9944-treated versus control retinas, respectively. However, comparing the di-22:6 PS species, there was nearly a 17-fold difference at 2 months and approximately sevenfold difference at 3 months between AY9944-treated and control retinas.

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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 to ESI-MS with direct infusion, essentially per the conditions given in legend, Fig. 1. Individual molecular species were quantified by comparisons to the internal standard as described in Experimental procedures. As a result of overlap with di-22:6 phosphatidylethanolamine, 18:0–22:6 PS was quantified using NL scanning of 87 amu, as indicated. Asterisks indicate statistical significance (*< 0.05, **< 0.005; Student's t-test, n = 3) for comparison of a given molecular species between AY9944-treated versus control rats at the same age.

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Figures 6a and b show the PE and plasmenylethanolamine molecular species, respectively, in the control and AY9944 treated rats. The major change in the retinal ethanolamine glycerophospholipid profile in the control rats over the first three postnatal months was the 2.4-fold increase in the mass of di-22:6 PE between 1 and 2 months, remaining high at 3 months. In contrast, retinal di-22:6 PE levels in AY9944-treated rats remained at the same levels at 2 and 3 months, compared with the first postnatal month. While no difference was observed at 1 month, there was a greater than threefold difference in the di-22:6 PE molecular species at 2 and 3 months, comparing AY9944-treated retinas to the corresponding age-matched controls. A 48–56% increase in the 18:0–22:6 PE molecular species was also observed as the control rats aged between one and three postnatal months (< 0.01 for comparisons between 1-month-old rats with either 2- or 3-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 2 and 3 months. Conversely, the levels of 18:0–22:4 PE were about twofold greater in retinas of AY9944-treated rats, compared with controls, at 2 and 3 months; however, while this change was statistically significant, this molecular species represents only a modest percentage of the total PE pool. It also should be noted that, comparing control versus AY9944-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 species enriched with DHA. Direct comparisons between the plasmalogen and diacyl molecular species of the ethanolamine glycerophospholipids are shown in Fig. 6c. At all ages of the untreated rats, plasmenylethanolamine molecular species represent ∼26–28% of the total retinal ethanolamine glycerophospholipid pool. In contrast, in AY9944-treated rats the plasmenylethanolamine molecular species represent 29%, 29%, and 32% of the retinal ethanolamine glycerophospholipid pool in 1-, 2-, and 3-month-old rats, respectively. This increased percentage of plasmenylethanolamine in the total pool is predominantly because of less PE molecular species present in the AY9944-treated rats compared with the untreated rats (Fig. 6c).

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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 with addition of ∼10 pmol NaOH per microliter and then subjected to ESI-MS with direct infusion, essentially per the conditions given in legend, Fig. 1. Individual phosphatidylethanolamine (PE) (a) and plasmenylethanolamine (b) molecular species were quantified by comparisons to the internal standard as described in Experimental procedures. As a result of overlap with 18:0–22:6 PS, di-22:6 PE was quantified using precursor ion scanning of 196 amu as indicated. The sum of total PE versus total plasmenylethanolamine molecular species is shown in (c). Statistical differences at *< 0.05 and **< 0.005 (Student’s t-test; n = 3) are indicated for comparison of a given molecular species between AY9944-treated and control rats at the same age.

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In comparison with differences in the PS and PE molecular species between AY9944-treated and control rat retinas, the differences in PC molecular species were considerably more complex (see Fig. 7a and b). 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 significantly lower in AY9944-treated rats than in controls at both 1 and 2 months age (Fig. 7a). Also, at all three ages examined, the disaturated PC molecular species (di-16:0 and 16:0–18:0) were significantly less in AY9944-treated rats, compared with age-matched controls. However, relative to controls, the difference in di-18:0 PC molecular species was statistically significant in AY9944-dependent only at 2 months. It also should be noted that, for these saturated PC molecular species, there was no age-dependent increase in the control rat retinas. However, in comparison with the content in 1-month-old control rats, the content of the retinal PC molecular species containing DHA (16:0–22:6, 18:0–22:6, and di-22:6) did increase, with statistical significance (< 0.005) in 2-month-old control rats and 18:0–22:6 and di-22:6 PC significantly (< 0.05) increased in 3-month-old rats (Fig. 7b). In striking contrast, there were no such increases in these DHA-containing molecular species in the retinas of AY9944-treated rats. These results in much larger age-matched differences in DHA-containing PC molecular species compared with the differences observed in saturated PC species (e.g. di-16:0 PC) in control versus AY9944-treated rats. In fact, as for PS and, to a lesser extent, for PE, the DHA content of PC molecular species was dramatically less in AY9944-treated rats in comparison with age-matched control rats at both 2 and 3 months: for 16:0–22:6, the difference was ∼2.5-fold and ∼1.9-fold, respectively; for 18:0–22:6, it was approximately twofold and ∼1.4-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 this molecular species at 2 and 3 months (approximately threefold and ∼1.9-fold, respectively) in AY9944-treated rats, compared with controls. Plasmenylcholine molecular species have previously been shown to represent a very minor percentage of the total retinal choline glycerophospholipid pool and are likely dispersed among multiple molecular species. Plasmenylcholine molecular species were not detectable under the direct infusion ESI-MS methods applied in these analyses. The plasmenylcholine molecular species likely represent < 1% of the TIC by ESI-MS detection, which limits their detection among many other ion species. In prior reports (reviewed in Fliesler and Anderson 1983), PC plasmalogen detection and analysis was facilitated by collapsing the species into one or two peaks (detection by gas chromatograph with flame ionization detection) upon converting the plasmalogen vinyl ethers into 16:0 and 18:0 dimethylacetal methanolysis products.

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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 with addition of ∼10 pmol NaOH per microliter and then subjected to ESI-MS with direct infusion, essentially per the conditions given in legend, Fig. 1, except in the positive ion mode. Individual molecular species including those without polyunsaturated fatty acids (a) and those containing polyunsaturated fatty acids (b) were quantified by comparisons to the internal standard as described in Experimental procedures. Statistical differences at *< 0.05 and **< 0.005 (Student’s t-test; n = 3) are indicated for comparison of a given molecular species between AY9944-treated and control rats at the same age.

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Analysis of serum and liver fatty acid composition in AY9944-treated and control rats

The results of the analysis of the total fatty acid composition of serum samples from AY9944-treated versus control rats as a function of postnatal age are shown in Fig. 8. Values for each acyl species are expressed as relative mole percent of the total. The predominant three fatty acids 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 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 acid deficiency in rats under the conditions employed. In fact, if anything, the levels of these fatty acids were slightly elevated in sera from the treated animals, relative to controls. At all three time points, the levels of arachidonic acid (20:4n6) in the treated animals were about half those of age-matched controls (< 0.001). At 1 and 2 months of treatment (but not at 3 months), there was a partially compensatory increase in the levels of 18:2n6 and 18:1 in treated animals, compared with controls (< 0.001).

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Figure 8.  Serum fatty acid composition of control (open bars) and AY9944-treated rats (filled bars) as a function of postnatal age; (a) 1 month; (b) 2 months; (c) 3 months. Total lipids were extracted and the corresponding fatty acid methyl esters (FAMEs) were prepared therefrom and analyzed by GLC (see Experimental procedures for details). Values are expressed as relative mol% (mean ± SD, n = 4). Statistical significance was evaluated using multivariant anova with a post hoc Scheffe test (*< 0.05; **< 0.01; ***< 0.001).

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The findings for serum were confirmed and extended by a similar analysis of liver fatty acid composition as a function of AY9944 treatment (Fig. 9). In this case, we examined tissues only from the 3-month time point, with the rationale that if AY9944 treatment caused a deficit in DHA or n3 fatty acids in general, it would be apparent in animals treated chronically for the longest period of time. Also, as the liver is the biogenic source of the DHA that is taken up by and incorporated into the retina (Scott and Bazan 1989), it would be the most likely place to observe such a deficit. However, as shown in Fig. 9, AY9944 treatment caused no loss of DHA in the livers of AY9944-treated rats, relative to age-matched controls on the same diet, even after 3 months of treatment. In fact, the livers of treated animals contained 1.34-fold more DHA than control livers (= 0.0285, n = 4). The levels of the dominant fatty acyl species, 20:4n6, in control livers were only modestly elevated (by 14.6%, = 0.0087, n = 4) relative to those of AY9944-treated rats. There were no statistically significant changes in the four other major 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.

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Figure 9.  Liver fatty acid composition of 3-month-old control (open bars) and AY9944-treated rats (filled bars). The treated rats received AY9944 throughout their entire lifetime. Total lipids were extracted and the corresponding fatty acid methyl esters (FAMEs) were prepared therefrom and analyzed by GLC (see Experimental procedures for details). Values are expressed as relative mol% (mean ± SD, n = 4). Statistical significance was evaluated using a two-tailed Student’s t-test (*< 0.05; **< 0.01; ***< 0.001).

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In the present study, we have demonstrated striking alterations in the phospholipid molecular species profile of the retina in rats treated with AY9944, compared with age- and sex-matched control rats, particularly with respect to the steady state levels of DHA. The predominant choline 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 2–3 months with AY9944 in comparison with age-matched control rats. The dominant DHA-containing retinal PE molecular species in both control and treated rats were 18:0–22:6 and di-22:6. In comparison with controls, AY9944 treatment resulted either in no difference (at 1 month), a 22% and 73% difference (by 2 months), or a 21% and 69% difference (by 3 months) of these ethanolamine glycerophospholipid molecular species, respectively. The predominant retinal PS molecular species were 18:0–22:6 and di-22:6; AY9944 treatment resulted in > 90% less di-22:6 PS, relative to controls. These findings demonstrate that the retinal lipidome is globally altered in the AY9944-treated rat, relative to control rats, over and above the changes in sterol composition (elevated 7DHC and reduced Chol levels) because of the primary inhibition of Chol biosynthesis caused by AY9944. To the extent that the AY9944-treated rat represents as suitable model of SLOS, these results suggest that additional metabolic compromise beyond the primary enzymatic defect in the Chol 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 has no reported effects on any enzyme involved in fatty acid or phospholipid biosynthesis. This is consistent with our finding that the n3 fatty acid content (especially DHA) of plasma and liver was not reduced in rats treated systemically with AY9944. Also, depletion of retinal DHA levels was achieved over a relatively brief time frame (2–3 months), even though the diet contained adequate levels of 18:3n3 (ca. 6 mol% of total fatty acids, as determined by GLC analysis; 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 that the vertebrate retina, particularly the outer segment membranes of retinal rod photoreceptor cells, and in striking contrast to other bodily tissues, strongly resists depletion of DHA and other n3 fatty acids when challenged with an n3-deficient diet (Futterman et al. 1971; Anderson and Maude 1972; Tinoco et al. 1977, 1978; Wiegand et al. 1991; Anderson et al. 1992; Bush et al. 1994). This is thought to be a consequence of an extremely active and efficient recycling mechanism, involving both the retinal pigment epithelium and the neural retina (Scott and Bazan 1989; Bazan et al. 1992, 1994).

The retina requires high levels of DHA for optimal function. Reduction of DHA in the rod outer segments (ROS) of rats (Benolken et al. 1973; Wheeler et al. 1975) and monkeys (Neuringer et al. 1984), by dietary restriction of n3 precursors, leads to reduced amplitudes and other changes in the electroretinogram. More recent studies have shown that ROS membranes with low DHA levels have a slower visual response to light (Niu et al. 2004). At the molecular level, this is seen as a slower transition of meta-rhodopsin-I to meta-rhodopsin-II, an important step in the activation of the G protein, transducin, and subsequent initiation of the visual signal. Studies in term and pre-term human infants have established that the development of visual acuity (Uauy et al. 1990, 1992; Birch et al. 1998) is dependent on a maternal supply of n3 fatty acids. Also, humans and dogs with inherited retinal degenerations have reduced plasma levels of n3 polyunsaturated fatty acids (Anderson et al. 1987, 1991). In addition, the retinas of dogs (Aguirre et al. 1997), rats (Anderson et al. 2002), and mice (Anderson et al. 2001a,b) with inherited retinal degenerations have significant reductions of DHA in their ROS membranes. These results are of further interest because the loss of DHA is not accompanied 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 this reduction in DHA, even in the presence of adequate levels of dietary n3 fatty acids (which we also observed in the current study) is unclear. The only other example of a reduction of DHA in the retina without a compensatory replacement by 22:5n6 occurs in albino rats (Penn and Anderson 1987, 1992) and mice (Káldi et al. 2003) raised in bright cyclic light. 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 photoreceptor cell membranes. This is consistent with what we observed in this animal model of SLOS. These changes are all the more intriguing because they reflect metabolic events occurring specifically in the retina, rather than those occurring in other tissues.

The mechanism by which this extraordinary remodeling of the retinal lipidome is achieved in SLOS rats likely involves multiple aspects of lipid transport and homeostasis, both systemic as well as retina-specific. First, it is possible that differential uptake of essential fatty acids may occur in the gut as a consequence of alterations in the composition and content of bile acids (required for solvation of lipids), because of reduced and abnormal sterol biosynthesis caused by AY9944. While not examined specifically in AY9944-treated rats, it has been reported that human SLOS patients have altered urinary bile acid composition, both with respect to decreased levels of normal bile acids and appearance of abnormal bile acids (Natowicz and Evans 1994). This would be expected, as Chol 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 cohort of SLOS patients, bile acid synthesis was not substantially different from control levels, and both normal primary and secondary bile acids were detected. Second, it is possible that the content of lipoprotein particles carrying DHA-enriched lipid constituents, which are assembled in and secreted by the liver, is different (i.e. DHA-deficient) in AY9944-treated versus control rats. This is an important consideration, as the retina derives its DHA from the liver, not from local de novo synthesis (Scott and Bazan 1989). However, the results of our detailed fatty acid analysis of serum and liver rule out this possibility, as serum DHA levels were equivalent to (at 1 and 2 months) or even slightly higher (at 3 months) in AY9944-treated animals, and liver DHA levels were slightly elevated at 3 months, compared with control values (see Figs 8 and 9). In addition to this prima facia lack of reduced DHA levels in serum and liver, systemic n3 fatty acid deficiency in AY9944-treated rats is also ruled out by the fact that there was no observed elevation of 22:5n6 in these tissues, which typically occurs in n3 fatty acid deficiency (see above). If fatty acid absorption in the gut (the first possibility mentioned above) was a significant factor, one would expect the fatty acid composition of serum and liver to be affected as well. However, given the fact that no DHA or more generalized n3 fatty acid deficiency was apparent in either serum or liver as a function of AY9944 treatment, the first possibility also tends to be ruled out. This leads to a third, and most likely, possibility: disruption in the uptake of blood-borne, DHA-containing lipoproteins by the retina, which also involves the retinal pigment epithelium at the choroidal interface, and subsequent intraretinal redistribution of the lipid constituents among the various retinal cell types and metabolic compartments, may be altered in AY9944-treated rats, compared with controls. This latter process is complex, involving multiple carriers (both intracellular and extracellular), receptors, and enzymes (Tserentsoodol et al. 2006). Fourth, there may be adaptive changes in de novo fatty acid synthesis in photoreceptors and other retinal cell types as a consequence of AY9944 treatment (see below). Resolution of these various possibilities is beyond the scope of the present study, but is the subject of active ongoing investigations in our laboratories.

Although DHA-containing phospholipid molecular species were the most profoundly altered lipids detected in this study, the levels of other fatty acids also were affected by AY9944 treatment. To some extent, this could reflect changes in de novo synthesis occurring within the retina per se. Although we have not yet performed metabolic labeling experiments to examine this further, we have obtained preliminary evidence, using microarray analysis, that indicates the expression levels of several genes involved in fatty acid and phospholipid biosynthesis are significantly altered in retinas of AY9944-treated rats, compared with controls, particularly after 2 months of treatment (Siddiqui et al. 2007). These include, but are not limited to, fatty acid synthase, stearoyl-CoA desaturase, acyl-CoA thioesterase, acyl-CoA synthetase, and CDP-diacylglycerol synthase. As AY9944 has no known direct effect on any of these enzymes, it is conceivable that crosstalk between the Chol biosynthetic pathway and the fatty acid and phospholipid biosynthetic pathways, mediated via sterol response elements and the sterol regulatory element-binding protein/SREBP cleavage-activating protein (SREBP/SCAP) system (reviewed in Shimano 2001; Rawson 2003; McPherson and Gauthier 2004), may explain, at least in part, these observations.

It should be appreciated that by 1 month of AY9944 treatment, there is no retinal degeneration and no electrophysiological dysfunction of the retina; yet, at the same time, retinal sterol 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 phospholipid molecular species after 1 month of AY9944 treatment were minimal. However, by 3 months of AY9944 treatment, substantial retinal degeneration and dysfunction accompanies even greater derangement of sterol metabolism and composition (Fliesler et al. 2004). Here, we found that retinal phospholipid molecular species composition is dramatically altered within 2–3 months of AY9944 treatment, compared with age-matched controls. Hence, phospholipid composition seems to correlate more closely with retina histological and electrophysiological integrity than does sterol composition in this animal model. However, it should not be inferred from these findings that reduced levels of DHA somehow caused the observed retinal degeneration. [Similarly, in the cases of hereditary retinal degeneration in animals, as mentioned above, there is no evidence that reduced levels of DHA cause the retinal degeneration.] A correlation between n3 fatty acid content and retinal development and function has been established for many years, both for rats (Benolken et al. 1973; Wheeler et al. 1975) as well as humans and non-human primates (Neuringer et al. 1984; Uauy et al. 1990, 1992; Birch et al. 1998). However, given the fact that we found no evidence for a systemic n3 fatty acid deficiency in this SLOS rat model, it is highly unlikely that feeding a high n3-containing (e.g. 18:3n3- or 22:6n3-rich) diet in addition to Chol supplementation, as opposed to a high-Chol diet alone, would provide any additional benefit with regard to improving retinal structure or function in the SLOS rat model. Indeed, even when AY9944-treated rats are fed a high-Chol diet for 2 months, which tends to nearly normalize their serum and retina sterol compositions, there is no protection against retinal degeneration (Fliesler et al. 2007). Furthermore, in a study by Martin et al. (2004), it was found that although supplementation with a diet enriched in n3 fatty acids was able to alter and partially restore the normal steady stated fatty acid profile of the retina (and, more specifically, of rod photoreceptor outer segment membranes), such dietary manipulation was neither able to protect against photoreceptor cell death nor alter the course of hereditary retinal degeneration in two different rat models carrying rhodopsin mutations linked to human retinal disease. In fact, the retina is known to conserve DHA under conditions of dietary n3 fatty acid deficiency (Anderson and Maude 1972; Wiegand et al. 1991), and even the small amounts of DHA present in standard rat chow are normally sufficient to support the high levels of DHA typically found in retinal photoreceptor membranes (Benolken et al. 1973). Taken together, these findings would suggest that the mechanism underlying the profound decrease in retinal DHA levels in the AY9944-treated rat model of SLOS is fundamentally different from that which 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 of SLOS patients may have altered fatty acid composition, particularly abnormally low DHA levels, compared with unaffected, age-matched normal individuals. Unfortunately, there are no reports in the literature, at present, to either confirm or negate this prediction, and obtaining SLOS 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, comparing them with normal controls, to ascertain if there is any correlation between disease severity and serum fatty acid profile. Our findings with the AY9944 rat model would predict a lack of such correlation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This 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 unrestricted departmental grants from Research to Prevent Blindness (SJF and REA). SJF is the recipient of a Research to Prevent Blindness Senior Scientific Investigator Award.

References

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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