Quantitative analysis of phospholipids containing arachidonate and docosahexaenoate chains in microdissected regions of mouse brain
2009; Elsevier BV; Volume: 51; Issue: 3 Linguagem: Inglês
10.1194/jlr.d001750
ISSN1539-7262
AutoresPaul H. Axelsen, Robert C. Murphy,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoPhospholipids containing polyunsaturated fatty acyl chains are prevalent among brain lipids, and regional differences in acyl chain distribution appear to have both functional and pathological significance. A method is described in which the combined application of GC and multiple reaction monitoring (MRM) MS yielded precise relative quantitation and approximate absolute quantitation of lipid species containing a particular fatty acyl chain in milligram-sized tissue samples. The method uses targeted MRM to identify specific molecular species of glycerophosphocholine lipids, glycerophospho-ethanolamine lipids, glycerophosphoinositol lipids, glycerophosphoserine lipids, glycero-phosphoglycerol lipids, and phosphatidic acids that contain esterified arachidonate (AA) and docosahexaenoate (DHA) separated during normal phase LC/MS/MS analysis. Quantitative analysis of the AA and DHA in the LC fractions is carried out using negative ion chemical ionization GC/MS and stable isotope dilution strategies. The method has been applied to assess the glycerophospholipid molecular species containing AA and DHA in microdissected samples of murine cerebral cortex and hippocampus. Results demonstrate the potential of this approach to identify regional differences in phospholipid concentration and reveal differences in specific phospholipid species between cortex and hippocampus. These differences may be related to the differential susceptibility of different brain regions to neurodegenerative disorders. Phospholipids containing polyunsaturated fatty acyl chains are prevalent among brain lipids, and regional differences in acyl chain distribution appear to have both functional and pathological significance. A method is described in which the combined application of GC and multiple reaction monitoring (MRM) MS yielded precise relative quantitation and approximate absolute quantitation of lipid species containing a particular fatty acyl chain in milligram-sized tissue samples. The method uses targeted MRM to identify specific molecular species of glycerophosphocholine lipids, glycerophospho-ethanolamine lipids, glycerophosphoinositol lipids, glycerophosphoserine lipids, glycero-phosphoglycerol lipids, and phosphatidic acids that contain esterified arachidonate (AA) and docosahexaenoate (DHA) separated during normal phase LC/MS/MS analysis. Quantitative analysis of the AA and DHA in the LC fractions is carried out using negative ion chemical ionization GC/MS and stable isotope dilution strategies. The method has been applied to assess the glycerophospholipid molecular species containing AA and DHA in microdissected samples of murine cerebral cortex and hippocampus. Results demonstrate the potential of this approach to identify regional differences in phospholipid concentration and reveal differences in specific phospholipid species between cortex and hippocampus. These differences may be related to the differential susceptibility of different brain regions to neurodegenerative disorders. ERRATAJournal of Lipid ResearchVol. 51Issue 5PreviewThe authors of the article "Quantitative analysis of phospholipids containing arachidonate and docosahexaenoate chains in microdissected regions of mouse brain" (J. Lipid Res. 51: 660–671) have advised the Journal that "docosahexaenoate" had been misspelled as "docosahexenoate". This misspelling appeared initially online but has since been corrected. Full-Text PDF Open Access Brain is the most lipid-rich tissue in mammals, and lipid metabolism disorders often have prominent neurological manifestations. 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Jayasinghe D.A. Ahiahonu P.W.K. Heath D. Yamazaki Y. Flax J. Krenitsky K.F. et al.Peripheral ethanolamine plasmalogen deficiency: a logical causative factor in Alzheimer's disease and dementia.J. Lipid Res. 2007; 48: 2485-2498Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). This study was undertaken to develop a micro-scale method for identifying and quantifying polyunsaturated phospholipid molecular species and to document regional differences in brain tissue. Emphasis was placed on identifying concentration differences in adjacent brain regions that differ in function and in their susceptibility to the neurodegenerative processes associated with oxidative stress in AD. The following synthetic phospholipid standards were obtained from Avanti Polar Lipids (Alabaster, AB): 17:0a/20:4a-phosphatidylcholine (PC), 17:0a/20:4a-PE, 17:0a/20:4a-phosphatidylglycerol (PG), 17:0a/20:4a-phosphatidylinositol (PI), 17:0a/20:4a-phosphatidylserine (PS), 17:0a/20:4a-phosphatidic acid (PA), 21:0a/22:6a-PC, 21:0a/22:6a-PE, 21:0a/22:6a-PG, 21:0a/22:6a-PI, 21:0a/22:6a-PS, and 21:0a/22:6a-PA in methanol at concentrations ranging from 10 to 30 μM, as well as (24:1)3/14:1- cardiolipin (CL), (14:1)3/15:1-CL, (15:0)3/16:1-CL, and (22:1)3/14:1-CL. Pentafluorobenzyl bromide and N,N-diisopropylethylamine were obtained from Sigma-Aldrich (St. Louis, MO). d8-AA and d5-DHA were obtained from Cayman Chemicals (Ann Arbor, MI). Neat AA and DHA for determining standard curves were obtained from NuCheck Prep Inc. (Elysian, MN). Three 9-month old 129S6/SVEV female mice (Taconic Farms, Inc., Hudson, NY) were euthanized by cervical dislocation. They were fed PMI type 5001 rodent chow ad libitum, and each mouse weighed approximately 20 g. Each brain was removed and frozen on dry ice within 5 min postmortem. Regions of interest were dissected from 1-mm-thick unstained coronal slices of mid- diencephalon while frozen under a dissecting microscope with the aid of a joystick micromanipulator (Eppendorf Transferman NK2, Westbury, NY). The two regions of interest in this work were the hippocampus (including dentate gyrus) and an adjacent portion of cerebral cortex of comparable size. One region was dissected from each brain hemisphere, yielding a total of six samples of hippocampus and six samples of cerebral cortex. Frozen tissue pieces were transferred to high-recovery clear borosilicate glass autosampler vials for weighing (9512S, Microsolv Technology Corp., Eatontown, NJ). The mass of the dissected tissue samples ranged from 2.0 to 4.5 mg, and they were stored at −80°C for up to 1 month before processing. Other than a brief period during weighing, tissue samples continuously remained frozen in liquid nitrogen (−196°C), dry ice (−78°C), or a freezer (−80°C) until extracted. Extraction was preceded by pulverizing the frozen samples in the bottom of the autosampler vial under liquid nitrogen with a Teflon pestle. Extraction was performed within the original vial using a modified Bligh-Dyer procedure. For tissue pieces from cerebral cortex and hippocampus (all weighing between 2.0 and 4.5 mg), a monophasic mixture of 400 μl methanol, 200 μl dichloromethane, and 160 μl of 5 mM ammonium acetate was added to the ground tissue, and the sample was sonicated with a tip sonicator for 60 s. Another 200 μl of dichloromethane and 160 μl of water were added to this monophasic homogenate, along with 10 μl/mg tissue of an internal standards mixture (see Table 1 for composition). This mixture was vortexed for 15 s, and the two resulting phases were clarified by brief low speed centrifugation. The lower phase (∼350 μl) was withdrawn and transferred to an autosampler vial with a PTFE lined cap (9532S, Microsolv Technology Corp., Eatontown, NJ) where it almost completely filled the vial. All extracts were kept at 5°C while in the autosampler awaiting analysis.TABLE 1Properties of the synthetic standards mixture, sn1/sn2 ratios, and extraction efficienciesAA StandardConcentration*The nominal concentration of each species, based on the manufacturer's label. (nM)sn1/sn2 RatioExtraction Efficiency (%)**Calculated as O/(A+O), where A and O are the transition signal recorded in the aqueous and organic phases, respectively.DHA StandardConcentration*The nominal concentration of each species, based on the manufacturer's label. (nM)sn1/sn2 RatioExtraction Efficiency (%)**Calculated as O/(A+O), where A and O are the transition signal recorded in the aqueous and organic phases, respectively.17:0/20:4-PC2210.37100.021:0/22:6-PC1730.3099.817:0/20:4-PE100.3888.121:0/22:6-PE490.3897.517:0/20:4-PG120.3399.621:0/22:6-PG100.3599.917:0/20:4-PI190.56100.021:0/22:6-PI160.5799.917:0/20:4-PS1072.2494.521:0/22:6-PS4382.2199.317:0/20:4-PA1133.15100.021:0/22:6-PA1173.0698.9* The nominal concentration of each species, based on the manufacturer's label.** Calculated as O/(A+O), where A and O are the transition signal recorded in the aqueous and organic phases, respectively. Open table in a new tab Aliquots (10 μl) of each extract were injected onto a 4.6 × 250 mm silica column (Rx-SIL, Agilent), through which solvents were pumped at 1 ml/min. Solvent A was 30 parts hexanes and 40 parts isopropanol; solvent B was 30 parts hexanes, 40 parts isopropanol, and 7 parts 11 mM ammonium acetate in water. Solvent B was increased linearly from 30% to 98% over 10 min and was held at 98% for 15 min. The column was reequilibrated with 30% solvent B for at least 5 min before another sample was injected. Column effluent was directed into a high precision flow splitter (Analytical Scientific Instruments, El Sobrante, CA) with precisely 25% of the effluent directed into the standard ESI source for LC/MS/MS analysis and 75% collected in fractions for GC/MS analysis. Two multiple reaction monitoring (MRM) analytical methods were developed using an ABI 4000 QTrap mass spectrometer (Toronto, Canada). One method monitored a set of collision-induced mass transitions in the negative ion mode corresponding to the production of AA anions (m/z 303.2) from various phospholipid and anions, and the 17:0 heptadacanoate anions (m/z 269.2) derived from sn1 chains in the AA-containing standards. The other method monitored the corresponding mass transitions for DHA anions (m/z 327.2) and 21:0 heneicosanoate anions (m/z 327.3). Each method divided the chromatographic separation into four periods, described below in detail. For all transitions, the dwell time was 100 ms, the source voltage was –4500 V, the collision voltage was –40 V, the collision gas was set to "medium", the resolution for both the first and third quadrupoles were set to "unit", and a drying gas at 300°C was applied to the spray. Transition peaks were integrated using Analyst 1.4.2 software, although all integrations were visually reviewed and many were adjusted manually. GC/MS was performed with a 30-m (30-m × 0.2-mm inner diameter × 0.25-µm film thickness) ZB-1 polydimethylsiloxane capillary gas chromatograph column (Phenomenex, Torrance, CA) attached to a ThermoFinnigan (San Jose, CA) Trace DSQ mass spectrometer. The injector temperature was maintained at 230°C, and the transfer line was kept at 290°C. The mass spectrometric experiments were performed in negative CI mode (70 eV) with a source temperature of 200°C. Helium was used as the carrier gas, with a constant flow rate of 0.8 ml/min. Phospholipid fractions for GC/MS analysis were saponified with 1 M NH4OH, mixed with d8-AA and d5-DHA internal standards, esterified with pentafluorobenzyl bromide in N,N-diisopropylethylamine, and extracted into isooctane as described previously (34Kayganich K. Murphy R.C. Molecular-species analysis of arachidonate containing glycerophosphocholines by tandem mass-spectrometry.J. Am. Soc. Mass Spectrom. 1991; 2: 45-54Crossref PubMed Scopus (39) Google Scholar). A standard curve for converting the internal standard signals to concentration was prepared with solutions of neat AA and DHA in methanol that were derivatized in the same manner. The integrated signal for each monitored mass transition was corrected for 13C content and processed as described in "Results." Quantitative sensitivity varied with the inherent difficulty of detecting lipid species in some headgroup classes and the amounts of these headgroup classes present in the brain. In addition, the results were subject to several assumptions that render them somewhat approximate. One assumption was that AA and DHA chains always occupied the sn2 position, as in the synthetic standards. However, lipids may have these chains in the sn1 position, they may have both an AA and a DHA chain, and they may even have two AA or two DHA chains. Precise corrections for these uncertainties are not available. When a lipid species has two AA or DHA chains, these species will be overcounted by a factor of 1 + r, where r is an sn1/sn2 ratio listed in Table 1. The numerical results provided for these species represent the measured values divided by this factor. No attempt was made to correct for differences in ionization efficiency due to differences in mass or due to differences between ether-linked and acyl-linked sn1 chains. Simple mass spectra of brain tissue extracts are illustrated in Fig. 1. The positive ion scans in both brain regions were dominated by the [M + H]+ ions of 16:0/18:1-PC (m/z 760.6), 16:0/16:0-PC (m/z 734.6), and 18:0/18:0-PC (m/z 790.6), as confirmed by separate MS/MS scans. The corresponding PE lipids were minor features of the spectrum (e.g., 16:0/18:1-PE at m/z 746.6). The most abundant PC lipids containing AA and DHA (e.g., 18:0/20:4-PC at m/z 810.6 and 18:0/22:6-PC at m/z 834.6) were also evident, but lipid species containing AA and DHA in any other headgroup classes could not be detected in the positive ion mode. The negative ion scans in both brain regions were dominated by the [M − H]− ions of 18:0/22:6-PS (m/z 834.5) and 18:0/20:4-PI (m/z 885.5). Some peaks, however, were most likely the superimposed ions of 18:0/22:6-PE and 18:0/18:0-PS, which were nearly isobaric at m/z 790.54 and 790.56, respectively. Differences between brain regions were discernable in these spectra, but relatively few lipids containing AA or DHA could be positively or uniquely identified, and quantitative reproducibility was poor at this level of mass spectrometric analysis. Chromatographic separation of most any type would help overcome these problems by delivering lipid species at characteristic elution times and reducing ion suppression. Normal phase separation on a silica column was chosen for further studies, because tissue extracts could be injected without further processing and lipid species with the same headgroup would elute at approximately the same times. A mixture of 12 phospholipid standards was examined to characterize the elution characteristics of the silica column and normal phase solvent system. Based on the elution times of the major headgroup classes, the chromatography system described above in "Methods" was divided into four periods (Fig. 2): PG standards eluted in period 1 between 7.0 and 9.5 min; PI and PE standards eluted in period 2 between 8.5 and 11.5 min; PA and PS standards eluted in period 3 between 11.5 and 13.5 min; and PC standards eluted in period 4 between 18 and 19 min. For PC standards, the yield of sn2 anions from [M + OAc−]− ions was slightly greater than from [M− CH3]− ions, so only the former were monitored. To assess extraction efficiency, aliquots of the standard mixture were subjected to the extraction procedure described above, and transition signals corresponding to each standard species were measured in both the upper (aqueous) and lower (organic) phase. Results suggested that extraction efficiency was >88% in all cases, and >99% in all but 4 cases (Table 1). The fatty acyl chains at the focus of this study, AA and DHA, are highly vulnerable to oxidative damage, so special precautions were taken to protect both synthetic standards and samples. These included grinding the samples under liquid nitrogen, the use of dicholoromethane instead of choloroform to avoid exposure of lipids to phosgene, and a minimum of solution transfers. Transitions 16 and 32 Da greater than that of the AA and DHA standards (i.e., the monooxidized and peroxidized products) were monitored in the synthetic standard mixture and in brain. Only insignificant trace amounts were found. To assess the suitability of the synthetic standards as internal standards for quantitative MS, a nominally equimolar mixture of the 12 standards was examined several times over a period of 4 weeks. This mixture was subjected to normal phase chromatography and MRM analysis as described above, and signal responses were corrected for 13C content. The fresh mixture yielded highly reproducible results and demonstrated that the AA-containing PE standard yielded 21.1-fold more signal than the AA-containing PI standard. Corresponding values for AA-containing PA and PS standards, as well as for the DHA-containing standards, are listed in Table 3.TABLE 3Sensitivity ratios for MS/MS detectionHeadgroup ClassesLipids Containing AALipids Containing DHAPE / PI21.161.7PA / PS3.36.1 Open table in a new tab Between analyses, the mixture was stored in plasma-cleaned autosampler vials at −80°C. After 4 weeks, the MRM transition signals were reexamined, and the signal responses from PG, PI, PE, PA, and PC standards were found to have decreased relative to the signals obtained for the PS standards. The losses ranged between 30% and 70%, with the greatest losses occurring in the PC standards. Losses of the PS standards were not assessed. Relative losses of similar magnitude were observed whether or not the vials had been plasma cleaned. The reason for these losses is not known but may involve the adherence of lipids in dilute solution to glass walls of the vial, glass-induced chemical decomposition, or oxidative damage. In any case, the losses precluded their use as reference standards. Nevertheless, they were useful as internal standards to correct for sample-sample variation. Extracts without added internal standards were initially examined with negative ion precursor scans for m/z 303.2 and m/z 327.3 ions to identify which molecular species containing AA and DHA, respectively, were present in the brain tissue, as well as to develop a list of mass spectral ion transitions for monitoring in the tandem mass spectrometer experiment. When a precursor ion peak eluted at a time characteristic of one headgroup and had a mass expected for a known phospholipid species bearing that headgroup, it was added to the list of monitored transitions for that headgroup class. The corresponding transition was also added to the list of monitored transitions for each of the other five headgroup classes. Ultimately, transitions for 27 individual species were monitored for each of the six AA-containing subclasses and for each of the six DHA-containing subclasses. Specific tandem mass spectrometric ion transitions are provided as supplementary Table I. Brain extracts without internal standards were also examined for naturally occurring species with the same mass transitions as the internal standards. None of the transitions involving the collision-induced production of 17:0 heptadecanoate or 21:0 heneicosanoate anions from internal standard parent ions yielded measurable signal, but some of the transitions corresponding to AA and DHA anions from internal standards yielded significant peaks. Presumably, these peaks arose from phospholipids containing O-linked 18:0 and 22:0 sn1 chains. Therefore, the expected signal for transitions yielding 20:4 and 22:6 chains from internal standards were calculated by dividing the signal obtained for transitions yielding 17:0 and 21:0 chains by the measured sn1/sn2 ratios listed in Table 1. The differences between the calculated signal for transitions yielding 20:4 and 22:6 chains, and the me
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