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Tandem Mass Spectrometry for Quantitative Analysis of Lipid Remodeling in Essential Fatty Acid Deficient Mice
Analytical Biochemistry 279, 179 –188 (2000) doi:10.1006/abio.1999.4452, available online at http://www.idealibrary.com on
Electrospray/Tandem Mass Spectrometry for Quantitative Analysis of Lipid Remodeling in Essential Fatty Acid Deficient Mice Kevin Duffin,* ,1 Mark Obukowicz,† Amiram Raz,‡ and J. J. Shieh* *Analytical Sciences Center, Monsanto Corporate Research, Monsanto Company, 700 Chesterfield Parkway North, St. Louis, Missouri 63198; †Discovery Pharmacology, G. D. Searle, St. Louis, Missouri 63198; and ‡Department of Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel
Received October 12, 1999
A method utilizing electrospray ionization coupled with tandem mass spectrometry was developed as a facile and rapid method to identify and quantify lipid remodeling in vivo. Electrospray/tandem mass spectrometric analyses were performed on lipids isolated from liver tissue and resident peritoneal cells from essential fatty acid sufficient and deficient mice. Essential fatty acid deficiency was chosen as the paradigm to evaluate the methodology because it epitomizes the most extreme dietary means of altering fatty acid composition of virtually all cellular lipid species. Qualitative and quantitative changes were measured in the phospholipid and cholesterol ester species directly in the chloroform/methanol lipid extract without any prior chromatographic separation. Lipid remodeling in liver and peritoneal cells from essential fatty acid deficient mice was qualitatively similar in cholesterol ester, phosphatidylcholine, and phosphatidylethanolamine. The monoenoic fatty acids palmitoleic acid (16:1 n-7) and oleic acid (18:1 n-9) were increased markedly, whereas all n-6 and n-3 polyunsaturated fatty acids were nearly depleted in phospholipid and cholesterol ester species. The n-9 polyunsaturated fatty acid surrogate, Mead acid (20:3 n-9), substituted for arachidonic acid (20:4 n-6) and docosahexaenoic acid (22:6 n-3) in phospholipid, but not in cholesterol ester, species. Another notable difference was that adrenic acid (22:4 n-6) and docosapentaenoic acid (22:5 n-6), both metabolites of arachidonic acid, accumulated in phospholipid and cholesterol ester species of peritoneal cells, but not in liver cells, of essential fatty acid sufficient mice. The overall body of data presented illustrates the imple-
1 To whom correspondence should be addressed. Fax: 314-7377099. E-mail: [email protected].
0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
mentation of electrospray/tandem mass spectrometry as a method for facile and direct quantification of changes in lipid species during lipid metabolic studies. © 2000 Academic Press Key Words: mass spectrometry; lipids; essential fatty acids; essential fatty acid deficient; remodeling.
Lipid remodeling of arachidonic acid is significant because of its pivotal role in inflammation. Reduction of arachidonic acid by dietary manipulation, either by n-3 polyunsaturated fatty acid (PUFA) 2 supplementation or by induction of essential fatty acid deficiency (EFAD), has been used to modulate the inflammatory response in several animal models (1–5). The reduction of arachidonic acid occurs in key phospholipid pools and leads to a decrease in the synthesis of proinflammatory eicosanoids, causing compromised macrophage and neutrophil function (4 –10). Studies with EFAD cells replete with exogenous fatty acids have shown that competition exists between proinflammatory arachidonic acid and competing anti-inflammatory PUFAs, for example, dihomo-␥-linolenic acid (DGLA; 20:3 n-6) and eicosapentaenoic acid (EPA; 20:5 n-3), for incorporation into cellular lipids (11, 12). However, analogous studies have not been performed in vivo, mainly due to the difficulty in facilely identifying and quantifying changes in molecular lipid species of various tissues or cells. Prior reports described the determination of fatty acid composition of triacylglycerides (TAG) and phos2 Abbreviations used: CE, cholesterol ester; EFAD, essential fatty acid deficient; ES/MS/MS, electrospray/tandem mass spectrometry; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; TAG, triacylglyceride.
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DUFFIN ET AL. TABLE 1
Fatty Acid Composition of the Corn Oil and EFAD Diets Content a (g/mg of diet) Fatty acid
Corn oil diet
EFAD diet
16:0 16:1 n-7 18:0 18:1 n-9 18:2 n-6 18:3 n-3 20:4 n-6 20:5 n-3 22:6 n-3 Total fatty acids
4.46 ⫾ 0.49 0.09 ⫾ 0.01 0.69 ⫾ 0.09 7.55 ⫾ 0.81 17.22 ⫾ 1.94 0.32 ⫾ 0.05 nd b nd nd 30.34 ⫾ 3.38
0.45 ⫾ 0.05 0.06 ⫾ 0.00 0.16 ⫾ 0.01 0.4 ⫾ 0.04 0.04 ⫾ 0.01 nd nd nd nd 1.10 ⫾ 0.09
a Amounts are expressed as micrograms of fatty acid per milligram of diet (average ⫾ SE, n ⫽ 3 replicate samples). b Not detectable.
lipid quantification in lipid remodeling studies in vivo. Sample preparation and analysis of phospholipid and CE species were simple and quick; tissue was homogenized, lipids were extracted in chloroform/methanol, and the lipid extract was then analyzed directly by ES/MS/MS without prior chromatographic separation. Lipid classes and individual fatty acids acylated to each lipid class were identified in complex mixtures by their distinctive fragmentation patterns. The methods described in this report can streamline lipid characterization by decreasing analysis time and by providing excellent sensitivity for identifying lipids present in low abundance in tissues, cells, or oils. Dietary studies on lipid remodeling using these methods can provide valuable insights into the physiological significance of fatty acid remodeling and may lead to a mechanismbased dietary treatment to prevent chronic inflammation or, possibly, cancer. MATERIALS AND METHODS
pholipid mixtures directly by mass spectrometry. Both negative- and positive-ion fast atom bombardment (13, 14), thermospray (15–17), and electrospray (18 –20) mass spectrometry have been used for lipid characterization. Tandem mass spectrometry proved useful for fragmenting lipid ions to determine their respective fatty acid composition. Previous reports showed that tandem mass spectrometry coupled with electrospray (ES/MS/MS) allowed selective identification and quantification of lipid species and their associated fatty acids in several different classes with minimal sample preparation (19, 21–25). However, there have been no ES/MS/MS methods described for identifying and quantifying cholesterol ester (CE) molecular species in tissue and body fluids. Generally, fatty acids acylated to cholesterol are identified and quantified by first separating CE from other lipid species and then saponifying, derivatizing, and separating the fatty acid derivatives by gas chromatography. An ES/MS/MS method for identifying and quantifying CE molecular species is described in this report. This methodology supplements existing methods for characterization of phospholipid molecular species classes and their cognate fatty acid components. The objective of this study was to demonstrate the utility of ES/MS/MS to readily and quickly identify and quantify changes in the molecular lipid species composition following dietary or therapeutic intervention. EFAD was chosen as the paradigm to evaluate the methodology because it epitomizes the most extreme dietary means of altering fatty acid composition of virtually all lipid species systemically. Liver and peritoneal cells were chosen for evaluation because they represent the “source” and “sink” of long-chain PUFA synthesis and deposition, respectively. The analysis described in this study is useful for CE and phospho-
Materials and Reagents Authentic lipids used as standards in ES/MS/MS method development and as internal standards for quantification were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL), Nu-Check Prep, Inc. (Elisa, MN), and Sigma Chemical Co. (St. Louis, MO). Organic solvents were Optima grade from Fisher Scientific (Pittsburgh, PA). Routine laboratory chemicals were purchased from Sigma Chemical Co. or Fisher Scientific. Sample preparation of lipids was performed in glass test tubes having Teflon-lined caps. Dietary Paradigm Female Balb/C mice were received as weanlings (3 weeks of age) and fed either a corn oil diet (AIN-76based corn oil diet; DYETS, Inc., Bethlehem, PA) or an EFAD diet (5803C, low essential fatty acid P.D.; Purina Test Diets, Richmond, IN) for a minimum of 8
TABLE 2
Precursor and Fragment Ions of Various Lipids Used in the Identification of Individual Lipid Classes Lipid
ES/MS/MS characteristic
Cholesterol ester Sphingomyelin Phosphatidylcholine Phosphatidylethanolamine Phosphatidylglycerol Phosphatidylserine Phosphatidylinositol Phosphatidic acid Cardiolipin
(M ⫹ NH 4) ⫹, m/z 369 fragment ion M ⫹, m/z 184 fragment ion M ⫹, m/z 184 fragment ion (M ⫹ H) ⫹, loss of 141 (M ⫹ NH 4) ⫹, loss of 172 and 189 (M ⫹ H) ⫹, loss of 185 (M ⫹ NH 4) ⫹, loss of 260 and 277 (M ⫹ NH 4) ⫹, loss of 98 and 115 (M ⫹ NH 4) ⫹, fragments to form diacylglycerol
TANDEM MASS SPECTROSCOPIC ANALYSIS OF LIPID REMODELING
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FIG. 1. Schematic of ES/MS/MS fragmentation. Phosphatidylcholine (A) and cholesterol ester (B) are shown as examples. Fragment ions at m/z 184 (A) and 369 (B) were the most abundant product ions of phosphatidylcholine and cholesterol esters, respectively, and were thus chosen for identification and quantification by parent ion ES/MS/MS. Analogous spectra of other phospholipids resulted in the selection of the major fragment ions used for identification and quantification (see Table 2). Note that the major point of fragmentation for all phospholipids occurred adjacent to the phosphate group.
weeks. The corn oil diet is essential fatty acid sufficient, being enriched in linoleic acid (LA; 18:2 n-6), whereas the EFAD diet is practically devoid of both n-6 and n-3 fatty acids (Table 1). Water and food were provided ad libitum throughout. A state of EFAD was confirmed by fatty acid composition analysis of liver tissue; the Mead acid (20:3 n-9)/arachidonic acid (20:4 n-6) ratio was approximately 5 at the end of the study (data not shown), much higher than the defined minimum value of 0.4 for EFAD (3). Lipid Preparation from Mouse Liver and Peritoneal Cells At the end of 8 weeks of feeding, the mice were sacrificed by CO 2 inhalation. Five milliliters of cold phosphate-buffered saline (PBS) was injected into the peritoneal cavity. After brief massaging to ensure mixing, the peritoneum was opened by a ventral incision and the wash fluid containing resident cells (primarily macrophages) was removed by aspiration. The wash fluid was centrifuged at 2000g to pellet the cells. The supernatant was aspirated carefully and the cell pellet was frozen immediately on dry ice. Liver tissue was then removed and blotted to remove blood. Liver tissue
samples were transferred to storage tubes and frozen immediately on dry ice. Both peritoneal cells and liver tissue were stored at ⫺80°C until lipid extraction. Total lipids from liver tissue or peritoneal cells were prepared according to a modified Bligh and Dyer (26) procedure. Liver tissue (100 mg) was added to 2 ml of CHCl 3/MeOH/H 2O (1:2:0.3) and homogenized with a handheld homogenizer (Tissue Tearor, Biospec Products, Inc., Bartlesville, OK) at room temperature. The homogenate was centrifuged at 10,000g for 5 min at room temperature. A second extraction was performed on the residual pellet by adding 1.2 ml of CHCl 3/ MeOH/H 2O (1:2:0.8), vortexing the residue vigorously, and then centrifuging at 10,000g for 5 min at room temperature. The supernatant was decanted and pooled with the first supernatant. The pooled supernatants were diluted with 0.9 ml of chloroform, yielding a final lipid extract from 100 mg of liver tissue solubilized in 4.1 ml of chloroform/methanol/water. Approximately 25 l of lipid extract was used for each ES/ MS/MS analysis, which corresponded to approximately 0.6 mg of liver tissue. For lipid extraction from peritoneal cells, 500 l of chloroform/methanol/water (1:2: 0.3) was added to the cell pellet. The mixture was
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vortexed and then centrifuged at 10,000g for 5 min at room temperature. A second extraction was performed on the cell pellet residue by adding 300 l of chloroform/methanol/water (1:2:0.8), vortexing vigorously, and then centrifuging at 10,000g for 5 min at room temperature. The supernatant was decanted and pooled with the first supernatant. The pooled supernatants were diluted with 250 l of chloroform, yielding a final lipid extract from peritoneal cells solubilized in 1.1 ml of chloroform/methanol/water. Approximately 25–50 l of cell lipid extract was used for each ES/ MS/MS analysis, which corresponded to approximately 10 5 cells. Electrospray and Tandem Mass Spectrometry For lipid quantification by ES/MS/MS, the liver or peritoneal cell lipid extract in chloroform/methanol/ water was added to an equal volume of chloroform/ methanol (1:1, v:v) containing 10 mM ammonium acetate and, in the case of the liver samples, an internal standard (10 g/ml final concentration). (The internal standard could have been added directly to liver tissue or peritoneal cells in order to compensate for lipid species more recalcitrant to the modified Bligh and Dyer extraction method [e.g., acidic and lysophospholipids].) Lipid extracts were introduced directly into a Sciex API III⫹ electrospray triple-quadrupole mass spectrometer (Sciex, Inc., Thornhill, ON) operated in the positive-ion mode. The lipid extracts were introduced continuously through the ion spray interface at a rate of 4 l/min. The ion spray interface was maintained at ⫹5 kV with respect to the ion entrance of the mass spectrometer. Air was used as the nebulizing gas. As lipid ions entered the mass spectrometer, they were accelerated through a potential difference of 50 V. This potential was sufficient to desolvate lipid ions, yet not sufficient to cause fragmentation of these ions. Mass analysis of sample ions was accomplished by scanning the first quadrupole of the triple-quadrupole mass spectrometer in 0.1-amu increments from 200 to 1200 amu in approximately 10 s. Mass resolution was set for complete baseline resolution of any ions differing by 1 amu. At least 10 individual scans were averaged to generate either precursor or neutral loss ES/MS/MS spectra that were used for quantification of individual lipid species. Product ion tandem mass spectra of lipid ions were used to identify the lipid species and the fatty acids acylated to a given lipid species. The spectra were acquired by passing the (M ⫹ NH 4) ⫹, (M ⫹ H) ⫹, or M ⫹ positive ions of intact, individual lipid species, which had been mass-selected with the first quadrupole, into the second quadrupole, where they were fragmented by collision with ultrapure argon gas. Fragment ions generated by these collisions then were mass-analyzed by
FIG. 2. Positive-ion tandem mass spectra of equimolar mixtures of phosphatidylcholine (A), phosphatidylethanolamine (B), and cholesterol ester (C). Approximately equal responses for each of the lipid species in each lipid class allowed direct quantification from ES/ MS/MS spectra without correction factors. Nonnatural internal standards (15:0/15:0-PC, 14:0/14:0-PE, or 15:0-CE) were spiked into each lipid extract to determine the exact quantities of individual lipid species.
the third quadrupole. The third quadrupole was scanned in 0.1-amu increments from 15 amu up to and including the mass of the parent ion in less than 10 s. At least 20 scans were averaged for each tandem mass spectrum. The argon target gas thickness was maintained at 2.3 ⫻ 10 14 atoms/cm 2 and a collision energy of 50 eV was chosen for this study. Mass spectrometric resolution of both precursor and product ions was set to achieve baseline resolution of ions that differed by 1 amu. Precursor ion and neutral loss tandem mass spectrometric analyses, which were used for detection and quantification of individual lipid species, were performed using the same instrument conditions as for the product ion ES/MS/MS analyses. Precursor ion ES/
TANDEM MASS SPECTROSCOPIC ANALYSIS OF LIPID REMODELING
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FIG. 3. Lipid species in phosphatidylcholine. Shown are representative ES/MS/MS tracings of lipid species from liver and peritoneal cells from essential fatty acid sufficient (A and B) and essential fatty acid deficient (C and D) mice.
MS/MS analyses were performed by setting the third quadrupole mass analyzer to pass a selected fragment ion. The first quadrupole then was scanned from 200 to 1200 amu to identify those lipids that dissociated to the prechosen fragment ion mass. Neutral loss ES/MS/MS experiments were performed by scanning the first and third quadrupole mass analyzers from 200 to 1200 amu simultaneously with a prechosen constant molecular weight offset. The molecular weights of lipids that dissociated by loss of a common neutral fragment were identified by this procedure. More than 50 individual scans were averaged to generate either precursor or neutral loss ES/MS/MS spectra that were used for quantification of individual lipid species. For all spectral scans, the standard error of the mean (SE) was ⬍5% of the mean within a given group (n ⫽ 5). RESULTS AND DISCUSSION
Validation of the ES/MS/MS Method for Lipid Analysis Previous reports have described the use of ES/ MS/MS to detect, resolve, and quantify PC, PE, PI, PS, sphingomyelin, and phosphatidylglycerol in complex lipid mixtures using selected precursor and neutral
loss scans (18 –20). In the present study, PC, PE, PS, PI, and CE were quantified in mouse liver and peritoneal cells using similar procedures with slight modifications. Ammonium acetate, which readily dissolves in methanol prior to dilution in chloroform, was added to the lipid extract just prior to sample analysis. The final composition of the lipid solvent for analysis was chloroform/methanol (2:1, v:v) containing 10 mM ammonium acetate. The addition of the ammonium salt allowed for facile ionization of all phospholipid and CE species in the positive-ion mode, and this mode was used throughout the study. Table 2 contains a summary of the phospholipid and CE precursor and fragment ions that were used for identification and, if spiked with an internal standard, quantification in this study. Qualitative and quantitative comparisons in liver and peritoneal cells from essential fatty acid sufficient and deficient groups were done for the major lipid classes, PC, PE, and CE. A schematic showing the ES/MS/MS fragmentation of PC and CE is shown in Fig. 1. As with PC, fragmentation of PE, PS, and PI occurs adjacent to the phosphate group. Equimolar standard mixtures of PC and PE were quantified by ES/MS/MS to determine whether any
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FIG. 4. Lipid species in phosphatidylethanolamine. Shown are representative ES/MS/MS tracings of lipid species from liver and peritoneal cells from essential fatty acid sufficient (A and B) and essential fatty acid deficient (C and D) mice.
differences in mass spectrometric responses existed. Biologically relevant standards of PC (Fig. 2A) and PE (Fig. 2B) containing fatty acids with different chain lengths and degrees of unsaturation were compared. Variation across lipid species was ⫾20%, whereas variation for a given lipid species compared to the internal standard was always ⬍5%. The relative ES/MS/MS response across different lipid classes was not determined. Thus, it is possible that there was a differential response of one lipid class relative to another. For example, it was noted that PC gave a much stronger mass spectrometric signal than other phospholipids, probably because it exists as an ion in solution. However, use of internal standards for each lipid class addressed any differential response that may have occurred. Cholesterol ester quantification by ES/MS/MS had not been described previously, so CE standards were first analyzed to determine their characteristic ionization and fragmentation patterns. Cholesterol esters readily ionized by ammonium attachment to yield (CE ⫹ NH 4) ⫹ when ammonium acetate was added to the chloroform/methanol solvent. Cholesterol esters fragmented by loss of the attached fatty acid to yield positively charged dehydrocholesterol at m/z 369 (Fig. 1B). This information was then used to design a precursor ion scan of the fragment ion at m/z 369 as a
method for selectively detecting the (M ⫹ NH 4) ⫹ ion of cholesterol ester species present in a lipid mixture. Application of this method to a solution of equimolar quantities of biologically relevant cholesterol ester standards yielded ES/MS/MS spectra with nearly equivalent CE responses (Fig. 2C), validating this method as a quantitative assay for CE species in lipid extracts. It should be noted, though, that no other accepted methods for cholesterol ester quantification were used to validate the values obtained with the ES/MS/MS method. Lipid Species Profiling in Liver and Peritoneal Cells from EFA-Sufficient vs EFAD Mice To illustrate the analytical utility of the technique developed, two types of comparisons are presented: (A) liver vs peritoneal cells of essential fatty acid sufficient (i.e., corn oil fed) mice and (B) liver vs peritoneal cells of essential fatty acid deficient mice. Qualitative comparisons of the lipid species in PC, PE, and CE are shown in the ES/MS/MS tracings (Figs. 3–5). Quantitative comparisons of the major PUFA species containing linoleic acid, arachidonic acid, docosahexaenoic acid, and Mead acid in PC and PE are presented in Table 3. Similar quantitative data for CE are shown in Fig. 6.
TANDEM MASS SPECTROSCOPIC ANALYSIS OF LIPID REMODELING
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FIG. 5. Lipid species in cholesterol ester. Shown are representative ES/MS/MS tracings of lipid species from liver and peritoneal cells from essential fatty acid sufficient (A and B) and essential fatty acid deficient (C and D) mice.
A. Differential Profile of Lipids in Liver and Peritoneal Cells Comparisons of lipid species from the livers and peritoneal cells of mice fed an essential fatty acid sufficient diet are shown for PC (Fig. 3A; Table 3), PE (Fig. 4A; Table 3), and CE (Figs. 5A and 6). The PC in peritoneal cells was especially rich in 16:0/16:0 and 16:0/18:1 species and also contains arachidonic acid (20:4 n-6) species as plasmalogens. Plasmalogen species, however, were not detected in PE of peritoneal cells, most of which are macrophages. It is not to be inferred that plasmalogen species do not exist in PE because they have been identified in PE of bovine brain (22). Significantly, docosahexaenoic acid (22:6 n-3) was present in PC and PE of liver, but was not detectable in peritoneal cell phospholipids. Peritoneal cells thus appeared to substitute longer chain n-6 PUFAs, namely, adrenic acid (22:4 n-6) and docosapentaenoic acid (22:5 n-6), for docosahexaenoic acid. A similar fatty acid pattern was also seen in the CE fraction of peritoneal cells, which was devoid of both docosahexaenoic acid and arachidonic acid but contained adrenic acid and docosapentaenoic acid. The enrichment of adrenic acid and docosapentaenoic acid in the CE and phospholipid lipid species of peritoneal cells (primarily macrophages) had
not been reported previously. Adrenic acid has been reported to be enriched in mouse kidney (27), human platelets (28), and rat pituitary (29, 30). Leukocytes have been described as possessing fatty acid elongase, but not fatty acid ⌬6 or ⌬5 desaturase activities (31– 34). Consequently, adrenic acid in peritoneal cells could be synthesized de novo by arachidonic acid elongation and/or obtained directly from the liver. In contrast, docosapentaenoic acid could not be synthesized de novo from arachidonic acid, but, instead, would most likely be obtained directly from the liver. The physiological significance of the enrichment of adrenic acid and docosapentaenoic acid in peritoneal cells is unknown. Both adrenic acid and docosapentaenoic acid could serve as a source of arachidonic acid via retroconversion (35). B. Lipid Remodeling Mediated by EFAD Comparison of the same tissues between mice fed an essential fatty acid sufficient or deficient diet revealed that there were general EFAD effects as well as tissuespecific EFAD effects. In general, EFAD led to the predominance of 16:0/18:1 and 18:1/18:1 as the major lipid species in both PC and PE of liver (Table 3; Figs. 3 and 4) and peritoneal cells (Figs. 3 and 4). These
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DUFFIN ET AL. TABLE 3
Quantification of Major Phosphatidylcholine and Phosphatidylethanolamine Lipid Species a Fatty acid 16:0
16:1 n-7
18:0
18:1 n-9
Total
A. Phosphatidylcholine species 18:2 n-6 species Corn oil EFAD 20:3 n-9 species Corn oil EFAD 20:4 n-6 species Corn oil EFAD 22:6 n-3 species Corn oil EFAD
2.7 ⫾ 0.1 0.14 ⫾ 0.01
nd b nd
1.3 ⫾ 0.0 nd
0.51 ⫾ 0.01 nd
4.5 0.14
nd 0.68 ⫾ 0.05
nd 0.06 ⫾ 0.01
nd 0.22 ⫾ 0.05
nd 0.30 ⫾ 0.07
nd 1.3
2.5 ⫾ 0.2 0.12 ⫾ 0.02
nd nd
1.9 ⫾ 0.2 0.30 ⫾ 0.07
0.97 ⫾ 0.08 0.10 ⫾ 0.04
5.4 0.52
1.6 ⫾ 0.0 0.05 ⫾ 0.01
nd nd
0.52 ⫾ 0.06 0.06 ⫾ 0.02
0.25 ⫾ 0.02 0.04 ⫾ 0.01
2.4 0.15
B. Phosphatidylethanolamine species 18:2 n-6 species Corn oil EFAD 20:3 n-9 species Corn oil EFAD 20:4 n-6 species Corn oil EFAD 22:6 n-3 species Corn oil EFAD
0.41 ⫾ 0.06 nd
nd b nd
0.62 ⫾ 0.06 nd
nd nd
1.2
nd 0.94 ⫾ 0.18
nd 0.10 ⫾ 0.01
nd 1.0 ⫾ 0.1
nd 1.8 ⫾ 0.1
nd 3.8
0.71 ⫾ 0.09 0.30 ⫾ 0.03
nd nd
2.3 ⫾ 0.3 0.35 ⫾ 0.03
1.9 ⫾ 0.3 0.69 ⫾ 0.09
4.9 1.3
3.8 ⫾ 0.4 0.58 ⫾ 0.03
nd nd
2.1 ⫾ 0.2 0.33 ⫾ 0.03
1.5 ⫾ 0.2 0.47 ⫾ 0.02
7.4 1.4
a A select subset of lipid species containing the key fatty acids, linoleic acid (18:2 n-6), Mead acid (20:3 n-9), arachidonic acid (20:4 n-6), and docosahexaenoic acid (22:6 n-3), was quantified in the livers of corn oil fed (i.e., essential fatty acid sufficient) and essential fatty acid deficient (EFAD) mice. The absolute amount of each phosphatidylcholine or phosphatidylethanolamine lipid species (g/mg of liver tissue) was determined by spiking a known amount of a nonbiological, phospholipid species into each sample as an internal standard. Results are expressed as mean ⫾ SE (n ⫽ 5 per group; two significant numbers). b Not detectable.
results clearly support past results showing induction and maintenance of ⌬9 desaturase activity consequential to the EFAD state (36). Another major difference in the fatty acid profile of liver and peritoneal cells from EFAD mice relates to the accumulation of Mead acid (20:3 n-9). In the liver, Mead acid was the major PUFA in all lipid classes, the major species being 16:0/20:3, 18:1/20:3, and 18:0/20:3 (Table 3; Figs. 3–5). In contrast, in peritoneal cells, there was no accumulation of Mead acid in CE and PE and only a trace amount in PC (Figs. 3– 6). These results showed the selectivity of Mead acid as an arachidonic acid surrogate in liver, but not in peritoneal, cells. The peritoneal cells, then, became depleted of arachidonic acid upon the advent of essential fatty acid deficiency, yet Mead acid did not substitute for arachidonic acid. The only major change in the peritoneal cells was the dramatic increase in monoenoic lipid species, 16:0/18:1, 18:1/18:1, and 16:1/ 16:1, in PC and PE (Figs. 3 and 4). Finally, the EFAD
FIG. 6. Composition of cholesterol ester species in the livers of essential fatty acid sufficient (i.e., corn oil fed) and essential fatty acid deficient mice.
TANDEM MASS SPECTROSCOPIC ANALYSIS OF LIPID REMODELING
condition in liver was uniquely characterized by the dramatic increase in the total CE content (essential fatty acid sufficient: 3.3 ⫾ 0.3 g/mg of liver; EFAD: 12.1 ⫾ 2.7 g/mg of liver). Particularly, there was enrichment in the CE content of the monoenoic fatty acids, palmitoleic acid (16:1 n-7), and, especially, oleic acid (18:1 n-9) (Fig. 6), suggesting induction of ⌬9 desaturase activity. A similar enrichment of monoenoic fatty acids in liver CE, as well as an overall increase in the content of liver CE, was also observed in mice dosed with a ⌬6 desaturase inhibitor (Duffin et al., submitted for publication). The dramatic increase in the abundance of monoenoic fatty acids suggested that the induction of ⌬9 desaturase activity was a compensatory response to the reduced availability of n-6 and n-3 long-chain PUFAs. The overall body of data presented illustrates the implementation of ES/MS/MS methodology to provide high resolution for the rapid and precise identification and quantification of molecular lipid species. Its utility lies in facile and direct quantification of changes (i.e., remodeling) in lipid species during lipid metabolic studies. REFERENCES 1. Reingold-Felsen, D., and Needleman, P. (1980) Eicosapentaenoic acid and the triene prostaglandins: Pharmacology and therapeutic potential. Trends Pharmacol. Sci. 1, 359 –361. 2. Galli, C., Marangoni, F., and Galella, G. (1993) Modulation of lipid derived mediators by polyunsaturated fatty acids. Prostaglandins Leukot. Essent. Fatty Acids 48, 51–55. 3. Holman, R. T. (1968) Essential fatty acid deficiency. in Progress in the Chemistry of Fats and Other Lipids (Holman, R. T., Ed.), Vol. 9, pp. 279 –348, Pergamon, London. 4. Lefkowith, J. B., Evers, A. S., Elliott, W. J., and Needleman, P. (1986) Essential fatty acid deficiency: A new look at an old problem. Prostaglandins Leukot. Med. 23, 123–127. 5. Lefkowith, J. B., Sprecher, H., and Needleman, P. (1986) The role and manipulation of eicosanoids in essential fatty acid deficiency. Prog. Lipid Res. 25, 111–117. 6. Lefkowith, J. B., Jakschik, B. A., Stahl, P., and Needleman, P. (1987) Metabolic and functional alterations in macrophages induced by essential fatty acid deficiency. J. Biol. Chem. 262, 6668 – 6675. 7. Lefkowith, J. B. (1988) Essential fatty acid deficiency inhibits the in vivo generation of leukotriene B 4 and suppresses levels of resident and elicited leukocytes in acute inflammation. J. Immunol. 140, 228 –233. 8. Wan, J. M.-F., Haw, M. P., and Blackburn, G. L. (1989) Nutrition, immune function, and inflammation: An overview. in Symposium on the Interaction between Nutrition and Inflammation, University of Southampton, Southampton, UK, September 8 –9, 1988 (Mathers, J. C., Quarterman, J., and Gurr, M. I., Eds.), Proceedings of the Nutrition Society, Vol. 48, pp. 315–335, Cambridge Univ. Press, Cambridge, UK. 9. Kinsella, J. E., Lokesh, B., Broughton, S., and Whelan, J. (1990) Dietary polyunsaturated fatty acids and eicosanoids. Potential effects on the modulation of inflammatory and immune cells: An overview. Nutrition 6, 24 – 44.
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10. Kinsella, J. E., and Lokesh, B. (1990) Dietary lipids, eicosanoids, and the immune system. Crit. Care Med. 18, S94 –S113. 11. Rubin, D., and Laposata, M. (1992) Cellular interactions between n-6 and n-3 fatty acids: A mass analysis of fatty acid elongation/desaturation, distribution among complex lipids, and conversion to eicosanoids. J. Lipid Res. 33, 1431–1440. 12. Rubin, D., and Laposata, M. (1991) Regulation of agonist-induced prostaglandin E1 versus prostaglandin E2 production: A mass analysis. J. Biol. Chem. 266, 23618 –23623. 13. Murphy, R. C., and Harrison, K. A. (1994) Fast-atom bombardment mass spectrometry of phospholipids. Mass Spectrom. Rev. 13, 57–75. 14. Kayganich-Harrison, K., and Murphy, R. C. (1994) Incorporation of stable isotope-labeled arachidonic acid into cellular phospholipid molecular species and analysis by fast atom bombardment tandem mass spectrometry. Biol. Mass Spectrom. 23, 562–571. 15. Ma, Y. C., and Kim, H. Y. (1995) Development of the online high-performance liquid chromatography/thermospray mass spectrometry method for the analysis of phospholipid molecular species in rat brain. Anal. Biochem. 226, 293–301. 16. Kuypers, F. A., Butikofer, P., and Shackleton, C. H. L. (1991) Application of liquid chromatography–thermospray mass spectrometry in the analysis of glycerophospholipid molecular species. J. Chromatogr. 562, 191–206. 17. Kim, H. Y., Yergey, J. A., and Salem, N. (1987) Determination of eicosanoids, phospholipids and related compounds by thermospray liquid chromatography–mass spectrometry. J. Chromatogr. 394, 155–170. 18. Hsu, F. F., Bohrer, A., and Turk, J. (1998) Formation of lithiated adducts of glycerophosphocholine lipids facilitates their identification by electrospray ionization tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 9, 516 –526. 19. Nakamura, T., Bratton, D. L., and Murphy, R. C. (1997) Analysis of epoxyeicosatrienoic and monohydroxyeicosatetraenoic acids esterified to phospholipids in human red blood cells by electrospray tandem mass spectrometry. J. Mass Spectrom. 32, 888 – 896. 20. Han, X., and Gross, R. W. (1994) Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids. Proc. Natl. Acad. Sci. USA 91, 10635–10639. 21. Duffin, K. L., Henion, J. D., and Shieh, J. J. (1991) Electrospray and tandem mass spectrometric characterization of acylglycerol mixtures that are dissolved in nonpolar solvents. Anal. Chem. 63, 1781–1788. 22. Kerwin, J. L., Tuininga, A. R., and Ericsson, L. H. (1994) Identification of molecular species of glycerophospholipids and sphingomyelin using electrospray mass spectrometry. J. Lipid Res. 35, 1102–1114. 23. Kerwin, J. L., Tuininga, A. R., Wiens, A. M., Wang, J. C., Torvik, J. J., Conrath, M. L., and MacKichan, J. K. (1995) Isoprenoidmediated changes in the glycerophospholipid molecular species of the sterol auxotrophic fungus Lagenidium giganteum. Microbiology 141, 399 – 410. 24. Karlsson, A. A., Michelsen, P., Larsen, A., and Odham, G. (1996) Normal-phase liquid chromatography class separation and species determination of phospholipids utilizing electrospray mass spectrometry/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 10, 775–780. 25. Ramanadham, S., Hsu, F.-F., Bohrer, A., Nowatzke, W., Ma, Z., and Turk, J. (1998) Electrospray ionization mass spectrometric analyses of phospholipids from rat and human pancreatic islets and subcellular membranes: Comparison to other tissues and implications for membrane fusion in insulin exocytosis. Biochemistry 37, 4553– 4567.
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26. Bligh, E. G., and Dyer, D. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. 27. Aukema, H. M., Yamaguchi, T., Takahashi, H., Celi, B., and Holub, B. J. (1992) Abnormal lipid and fatty acid compositions of kidneys from mice with polycystic kidney disease. Lipids 27, 429 – 435. 28. Sanders, T. A., and Roshanai, F. (1992) Platelet phospholipid fatty acid composition and function in vegans compared with age- and sex-matched omnivore controls. Eur. J. Clin. Nutr. 46, 823– 831. 29. Alessio, M. L., Wandscheer, D. E., Soares, M. C., Clauser, H., Enjalbert, A., Kordon, C., and Leger, C. L. (1992) Effect of an essential fatty acid deficiency on the phospholipid composition in anterior pituitary membranes. Biochem. Biophys. Res. Commun. 183, 1047–1055. 30. Soares, M. C., Alessio, M. L., Leger, C. L., Bluet-Pajot, M. T., Clauser, H., Enjalbert, A., Kordon, C., and Wandscheer, D. E. (1995) Effect of essential fatty acid deficiency on membrane fatty acid content and growth hormone stimulation of rat pituitaries during post-natal development. J. Lipid Res. 36, 1401–1406. 31. Chapkin, R. S., Somers, S. D., and Erickson, K. L. (1988) Inability of murine peritoneal macrophages to convert linoleic acid into
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Electrospray/Tandem Mass Spectrometry for Quantitative Analysis of Lipid Remodeling in Essential Fatty Acid Deficient Mice Kevin Duffin,* ,1 Mark Obukowicz,† Amiram Raz,‡ and J. J. Shieh* *Analytical Sciences Center, Monsanto Corporate Research, Monsanto Company, 700 Chesterfield Parkway North, St. Louis, Missouri 63198; †Discovery Pharmacology, G. D. Searle, St. Louis, Missouri 63198; and ‡Department of Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel
Received October 12, 1999
A method utilizing electrospray ionization coupled with tandem mass spectrometry was developed as a facile and rapid method to identify and quantify lipid remodeling in vivo. Electrospray/tandem mass spectrometric analyses were performed on lipids isolated from liver tissue and resident peritoneal cells from essential fatty acid sufficient and deficient mice. Essential fatty acid deficiency was chosen as the paradigm to evaluate the methodology because it epitomizes the most extreme dietary means of altering fatty acid composition of virtually all cellular lipid species. Qualitative and quantitative changes were measured in the phospholipid and cholesterol ester species directly in the chloroform/methanol lipid extract without any prior chromatographic separation. Lipid remodeling in liver and peritoneal cells from essential fatty acid deficient mice was qualitatively similar in cholesterol ester, phosphatidylcholine, and phosphatidylethanolamine. The monoenoic fatty acids palmitoleic acid (16:1 n-7) and oleic acid (18:1 n-9) were increased markedly, whereas all n-6 and n-3 polyunsaturated fatty acids were nearly depleted in phospholipid and cholesterol ester species. The n-9 polyunsaturated fatty acid surrogate, Mead acid (20:3 n-9), substituted for arachidonic acid (20:4 n-6) and docosahexaenoic acid (22:6 n-3) in phospholipid, but not in cholesterol ester, species. Another notable difference was that adrenic acid (22:4 n-6) and docosapentaenoic acid (22:5 n-6), both metabolites of arachidonic acid, accumulated in phospholipid and cholesterol ester species of peritoneal cells, but not in liver cells, of essential fatty acid sufficient mice. The overall body of data presented illustrates the imple-
1 To whom correspondence should be addressed. Fax: 314-7377099. E-mail: [email protected].
0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
mentation of electrospray/tandem mass spectrometry as a method for facile and direct quantification of changes in lipid species during lipid metabolic studies. © 2000 Academic Press Key Words: mass spectrometry; lipids; essential fatty acids; essential fatty acid deficient; remodeling.
Lipid remodeling of arachidonic acid is significant because of its pivotal role in inflammation. Reduction of arachidonic acid by dietary manipulation, either by n-3 polyunsaturated fatty acid (PUFA) 2 supplementation or by induction of essential fatty acid deficiency (EFAD), has been used to modulate the inflammatory response in several animal models (1–5). The reduction of arachidonic acid occurs in key phospholipid pools and leads to a decrease in the synthesis of proinflammatory eicosanoids, causing compromised macrophage and neutrophil function (4 –10). Studies with EFAD cells replete with exogenous fatty acids have shown that competition exists between proinflammatory arachidonic acid and competing anti-inflammatory PUFAs, for example, dihomo-␥-linolenic acid (DGLA; 20:3 n-6) and eicosapentaenoic acid (EPA; 20:5 n-3), for incorporation into cellular lipids (11, 12). However, analogous studies have not been performed in vivo, mainly due to the difficulty in facilely identifying and quantifying changes in molecular lipid species of various tissues or cells. Prior reports described the determination of fatty acid composition of triacylglycerides (TAG) and phos2 Abbreviations used: CE, cholesterol ester; EFAD, essential fatty acid deficient; ES/MS/MS, electrospray/tandem mass spectrometry; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; TAG, triacylglyceride.
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Fatty Acid Composition of the Corn Oil and EFAD Diets Content a (g/mg of diet) Fatty acid
Corn oil diet
EFAD diet
16:0 16:1 n-7 18:0 18:1 n-9 18:2 n-6 18:3 n-3 20:4 n-6 20:5 n-3 22:6 n-3 Total fatty acids
4.46 ⫾ 0.49 0.09 ⫾ 0.01 0.69 ⫾ 0.09 7.55 ⫾ 0.81 17.22 ⫾ 1.94 0.32 ⫾ 0.05 nd b nd nd 30.34 ⫾ 3.38
0.45 ⫾ 0.05 0.06 ⫾ 0.00 0.16 ⫾ 0.01 0.4 ⫾ 0.04 0.04 ⫾ 0.01 nd nd nd nd 1.10 ⫾ 0.09
a Amounts are expressed as micrograms of fatty acid per milligram of diet (average ⫾ SE, n ⫽ 3 replicate samples). b Not detectable.
lipid quantification in lipid remodeling studies in vivo. Sample preparation and analysis of phospholipid and CE species were simple and quick; tissue was homogenized, lipids were extracted in chloroform/methanol, and the lipid extract was then analyzed directly by ES/MS/MS without prior chromatographic separation. Lipid classes and individual fatty acids acylated to each lipid class were identified in complex mixtures by their distinctive fragmentation patterns. The methods described in this report can streamline lipid characterization by decreasing analysis time and by providing excellent sensitivity for identifying lipids present in low abundance in tissues, cells, or oils. Dietary studies on lipid remodeling using these methods can provide valuable insights into the physiological significance of fatty acid remodeling and may lead to a mechanismbased dietary treatment to prevent chronic inflammation or, possibly, cancer. MATERIALS AND METHODS
pholipid mixtures directly by mass spectrometry. Both negative- and positive-ion fast atom bombardment (13, 14), thermospray (15–17), and electrospray (18 –20) mass spectrometry have been used for lipid characterization. Tandem mass spectrometry proved useful for fragmenting lipid ions to determine their respective fatty acid composition. Previous reports showed that tandem mass spectrometry coupled with electrospray (ES/MS/MS) allowed selective identification and quantification of lipid species and their associated fatty acids in several different classes with minimal sample preparation (19, 21–25). However, there have been no ES/MS/MS methods described for identifying and quantifying cholesterol ester (CE) molecular species in tissue and body fluids. Generally, fatty acids acylated to cholesterol are identified and quantified by first separating CE from other lipid species and then saponifying, derivatizing, and separating the fatty acid derivatives by gas chromatography. An ES/MS/MS method for identifying and quantifying CE molecular species is described in this report. This methodology supplements existing methods for characterization of phospholipid molecular species classes and their cognate fatty acid components. The objective of this study was to demonstrate the utility of ES/MS/MS to readily and quickly identify and quantify changes in the molecular lipid species composition following dietary or therapeutic intervention. EFAD was chosen as the paradigm to evaluate the methodology because it epitomizes the most extreme dietary means of altering fatty acid composition of virtually all lipid species systemically. Liver and peritoneal cells were chosen for evaluation because they represent the “source” and “sink” of long-chain PUFA synthesis and deposition, respectively. The analysis described in this study is useful for CE and phospho-
Materials and Reagents Authentic lipids used as standards in ES/MS/MS method development and as internal standards for quantification were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL), Nu-Check Prep, Inc. (Elisa, MN), and Sigma Chemical Co. (St. Louis, MO). Organic solvents were Optima grade from Fisher Scientific (Pittsburgh, PA). Routine laboratory chemicals were purchased from Sigma Chemical Co. or Fisher Scientific. Sample preparation of lipids was performed in glass test tubes having Teflon-lined caps. Dietary Paradigm Female Balb/C mice were received as weanlings (3 weeks of age) and fed either a corn oil diet (AIN-76based corn oil diet; DYETS, Inc., Bethlehem, PA) or an EFAD diet (5803C, low essential fatty acid P.D.; Purina Test Diets, Richmond, IN) for a minimum of 8
TABLE 2
Precursor and Fragment Ions of Various Lipids Used in the Identification of Individual Lipid Classes Lipid
ES/MS/MS characteristic
Cholesterol ester Sphingomyelin Phosphatidylcholine Phosphatidylethanolamine Phosphatidylglycerol Phosphatidylserine Phosphatidylinositol Phosphatidic acid Cardiolipin
(M ⫹ NH 4) ⫹, m/z 369 fragment ion M ⫹, m/z 184 fragment ion M ⫹, m/z 184 fragment ion (M ⫹ H) ⫹, loss of 141 (M ⫹ NH 4) ⫹, loss of 172 and 189 (M ⫹ H) ⫹, loss of 185 (M ⫹ NH 4) ⫹, loss of 260 and 277 (M ⫹ NH 4) ⫹, loss of 98 and 115 (M ⫹ NH 4) ⫹, fragments to form diacylglycerol
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FIG. 1. Schematic of ES/MS/MS fragmentation. Phosphatidylcholine (A) and cholesterol ester (B) are shown as examples. Fragment ions at m/z 184 (A) and 369 (B) were the most abundant product ions of phosphatidylcholine and cholesterol esters, respectively, and were thus chosen for identification and quantification by parent ion ES/MS/MS. Analogous spectra of other phospholipids resulted in the selection of the major fragment ions used for identification and quantification (see Table 2). Note that the major point of fragmentation for all phospholipids occurred adjacent to the phosphate group.
weeks. The corn oil diet is essential fatty acid sufficient, being enriched in linoleic acid (LA; 18:2 n-6), whereas the EFAD diet is practically devoid of both n-6 and n-3 fatty acids (Table 1). Water and food were provided ad libitum throughout. A state of EFAD was confirmed by fatty acid composition analysis of liver tissue; the Mead acid (20:3 n-9)/arachidonic acid (20:4 n-6) ratio was approximately 5 at the end of the study (data not shown), much higher than the defined minimum value of 0.4 for EFAD (3). Lipid Preparation from Mouse Liver and Peritoneal Cells At the end of 8 weeks of feeding, the mice were sacrificed by CO 2 inhalation. Five milliliters of cold phosphate-buffered saline (PBS) was injected into the peritoneal cavity. After brief massaging to ensure mixing, the peritoneum was opened by a ventral incision and the wash fluid containing resident cells (primarily macrophages) was removed by aspiration. The wash fluid was centrifuged at 2000g to pellet the cells. The supernatant was aspirated carefully and the cell pellet was frozen immediately on dry ice. Liver tissue was then removed and blotted to remove blood. Liver tissue
samples were transferred to storage tubes and frozen immediately on dry ice. Both peritoneal cells and liver tissue were stored at ⫺80°C until lipid extraction. Total lipids from liver tissue or peritoneal cells were prepared according to a modified Bligh and Dyer (26) procedure. Liver tissue (100 mg) was added to 2 ml of CHCl 3/MeOH/H 2O (1:2:0.3) and homogenized with a handheld homogenizer (Tissue Tearor, Biospec Products, Inc., Bartlesville, OK) at room temperature. The homogenate was centrifuged at 10,000g for 5 min at room temperature. A second extraction was performed on the residual pellet by adding 1.2 ml of CHCl 3/ MeOH/H 2O (1:2:0.8), vortexing the residue vigorously, and then centrifuging at 10,000g for 5 min at room temperature. The supernatant was decanted and pooled with the first supernatant. The pooled supernatants were diluted with 0.9 ml of chloroform, yielding a final lipid extract from 100 mg of liver tissue solubilized in 4.1 ml of chloroform/methanol/water. Approximately 25 l of lipid extract was used for each ES/ MS/MS analysis, which corresponded to approximately 0.6 mg of liver tissue. For lipid extraction from peritoneal cells, 500 l of chloroform/methanol/water (1:2: 0.3) was added to the cell pellet. The mixture was
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vortexed and then centrifuged at 10,000g for 5 min at room temperature. A second extraction was performed on the cell pellet residue by adding 300 l of chloroform/methanol/water (1:2:0.8), vortexing vigorously, and then centrifuging at 10,000g for 5 min at room temperature. The supernatant was decanted and pooled with the first supernatant. The pooled supernatants were diluted with 250 l of chloroform, yielding a final lipid extract from peritoneal cells solubilized in 1.1 ml of chloroform/methanol/water. Approximately 25–50 l of cell lipid extract was used for each ES/ MS/MS analysis, which corresponded to approximately 10 5 cells. Electrospray and Tandem Mass Spectrometry For lipid quantification by ES/MS/MS, the liver or peritoneal cell lipid extract in chloroform/methanol/ water was added to an equal volume of chloroform/ methanol (1:1, v:v) containing 10 mM ammonium acetate and, in the case of the liver samples, an internal standard (10 g/ml final concentration). (The internal standard could have been added directly to liver tissue or peritoneal cells in order to compensate for lipid species more recalcitrant to the modified Bligh and Dyer extraction method [e.g., acidic and lysophospholipids].) Lipid extracts were introduced directly into a Sciex API III⫹ electrospray triple-quadrupole mass spectrometer (Sciex, Inc., Thornhill, ON) operated in the positive-ion mode. The lipid extracts were introduced continuously through the ion spray interface at a rate of 4 l/min. The ion spray interface was maintained at ⫹5 kV with respect to the ion entrance of the mass spectrometer. Air was used as the nebulizing gas. As lipid ions entered the mass spectrometer, they were accelerated through a potential difference of 50 V. This potential was sufficient to desolvate lipid ions, yet not sufficient to cause fragmentation of these ions. Mass analysis of sample ions was accomplished by scanning the first quadrupole of the triple-quadrupole mass spectrometer in 0.1-amu increments from 200 to 1200 amu in approximately 10 s. Mass resolution was set for complete baseline resolution of any ions differing by 1 amu. At least 10 individual scans were averaged to generate either precursor or neutral loss ES/MS/MS spectra that were used for quantification of individual lipid species. Product ion tandem mass spectra of lipid ions were used to identify the lipid species and the fatty acids acylated to a given lipid species. The spectra were acquired by passing the (M ⫹ NH 4) ⫹, (M ⫹ H) ⫹, or M ⫹ positive ions of intact, individual lipid species, which had been mass-selected with the first quadrupole, into the second quadrupole, where they were fragmented by collision with ultrapure argon gas. Fragment ions generated by these collisions then were mass-analyzed by
FIG. 2. Positive-ion tandem mass spectra of equimolar mixtures of phosphatidylcholine (A), phosphatidylethanolamine (B), and cholesterol ester (C). Approximately equal responses for each of the lipid species in each lipid class allowed direct quantification from ES/ MS/MS spectra without correction factors. Nonnatural internal standards (15:0/15:0-PC, 14:0/14:0-PE, or 15:0-CE) were spiked into each lipid extract to determine the exact quantities of individual lipid species.
the third quadrupole. The third quadrupole was scanned in 0.1-amu increments from 15 amu up to and including the mass of the parent ion in less than 10 s. At least 20 scans were averaged for each tandem mass spectrum. The argon target gas thickness was maintained at 2.3 ⫻ 10 14 atoms/cm 2 and a collision energy of 50 eV was chosen for this study. Mass spectrometric resolution of both precursor and product ions was set to achieve baseline resolution of ions that differed by 1 amu. Precursor ion and neutral loss tandem mass spectrometric analyses, which were used for detection and quantification of individual lipid species, were performed using the same instrument conditions as for the product ion ES/MS/MS analyses. Precursor ion ES/
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FIG. 3. Lipid species in phosphatidylcholine. Shown are representative ES/MS/MS tracings of lipid species from liver and peritoneal cells from essential fatty acid sufficient (A and B) and essential fatty acid deficient (C and D) mice.
MS/MS analyses were performed by setting the third quadrupole mass analyzer to pass a selected fragment ion. The first quadrupole then was scanned from 200 to 1200 amu to identify those lipids that dissociated to the prechosen fragment ion mass. Neutral loss ES/MS/MS experiments were performed by scanning the first and third quadrupole mass analyzers from 200 to 1200 amu simultaneously with a prechosen constant molecular weight offset. The molecular weights of lipids that dissociated by loss of a common neutral fragment were identified by this procedure. More than 50 individual scans were averaged to generate either precursor or neutral loss ES/MS/MS spectra that were used for quantification of individual lipid species. For all spectral scans, the standard error of the mean (SE) was ⬍5% of the mean within a given group (n ⫽ 5). RESULTS AND DISCUSSION
Validation of the ES/MS/MS Method for Lipid Analysis Previous reports have described the use of ES/ MS/MS to detect, resolve, and quantify PC, PE, PI, PS, sphingomyelin, and phosphatidylglycerol in complex lipid mixtures using selected precursor and neutral
loss scans (18 –20). In the present study, PC, PE, PS, PI, and CE were quantified in mouse liver and peritoneal cells using similar procedures with slight modifications. Ammonium acetate, which readily dissolves in methanol prior to dilution in chloroform, was added to the lipid extract just prior to sample analysis. The final composition of the lipid solvent for analysis was chloroform/methanol (2:1, v:v) containing 10 mM ammonium acetate. The addition of the ammonium salt allowed for facile ionization of all phospholipid and CE species in the positive-ion mode, and this mode was used throughout the study. Table 2 contains a summary of the phospholipid and CE precursor and fragment ions that were used for identification and, if spiked with an internal standard, quantification in this study. Qualitative and quantitative comparisons in liver and peritoneal cells from essential fatty acid sufficient and deficient groups were done for the major lipid classes, PC, PE, and CE. A schematic showing the ES/MS/MS fragmentation of PC and CE is shown in Fig. 1. As with PC, fragmentation of PE, PS, and PI occurs adjacent to the phosphate group. Equimolar standard mixtures of PC and PE were quantified by ES/MS/MS to determine whether any
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FIG. 4. Lipid species in phosphatidylethanolamine. Shown are representative ES/MS/MS tracings of lipid species from liver and peritoneal cells from essential fatty acid sufficient (A and B) and essential fatty acid deficient (C and D) mice.
differences in mass spectrometric responses existed. Biologically relevant standards of PC (Fig. 2A) and PE (Fig. 2B) containing fatty acids with different chain lengths and degrees of unsaturation were compared. Variation across lipid species was ⫾20%, whereas variation for a given lipid species compared to the internal standard was always ⬍5%. The relative ES/MS/MS response across different lipid classes was not determined. Thus, it is possible that there was a differential response of one lipid class relative to another. For example, it was noted that PC gave a much stronger mass spectrometric signal than other phospholipids, probably because it exists as an ion in solution. However, use of internal standards for each lipid class addressed any differential response that may have occurred. Cholesterol ester quantification by ES/MS/MS had not been described previously, so CE standards were first analyzed to determine their characteristic ionization and fragmentation patterns. Cholesterol esters readily ionized by ammonium attachment to yield (CE ⫹ NH 4) ⫹ when ammonium acetate was added to the chloroform/methanol solvent. Cholesterol esters fragmented by loss of the attached fatty acid to yield positively charged dehydrocholesterol at m/z 369 (Fig. 1B). This information was then used to design a precursor ion scan of the fragment ion at m/z 369 as a
method for selectively detecting the (M ⫹ NH 4) ⫹ ion of cholesterol ester species present in a lipid mixture. Application of this method to a solution of equimolar quantities of biologically relevant cholesterol ester standards yielded ES/MS/MS spectra with nearly equivalent CE responses (Fig. 2C), validating this method as a quantitative assay for CE species in lipid extracts. It should be noted, though, that no other accepted methods for cholesterol ester quantification were used to validate the values obtained with the ES/MS/MS method. Lipid Species Profiling in Liver and Peritoneal Cells from EFA-Sufficient vs EFAD Mice To illustrate the analytical utility of the technique developed, two types of comparisons are presented: (A) liver vs peritoneal cells of essential fatty acid sufficient (i.e., corn oil fed) mice and (B) liver vs peritoneal cells of essential fatty acid deficient mice. Qualitative comparisons of the lipid species in PC, PE, and CE are shown in the ES/MS/MS tracings (Figs. 3–5). Quantitative comparisons of the major PUFA species containing linoleic acid, arachidonic acid, docosahexaenoic acid, and Mead acid in PC and PE are presented in Table 3. Similar quantitative data for CE are shown in Fig. 6.
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FIG. 5. Lipid species in cholesterol ester. Shown are representative ES/MS/MS tracings of lipid species from liver and peritoneal cells from essential fatty acid sufficient (A and B) and essential fatty acid deficient (C and D) mice.
A. Differential Profile of Lipids in Liver and Peritoneal Cells Comparisons of lipid species from the livers and peritoneal cells of mice fed an essential fatty acid sufficient diet are shown for PC (Fig. 3A; Table 3), PE (Fig. 4A; Table 3), and CE (Figs. 5A and 6). The PC in peritoneal cells was especially rich in 16:0/16:0 and 16:0/18:1 species and also contains arachidonic acid (20:4 n-6) species as plasmalogens. Plasmalogen species, however, were not detected in PE of peritoneal cells, most of which are macrophages. It is not to be inferred that plasmalogen species do not exist in PE because they have been identified in PE of bovine brain (22). Significantly, docosahexaenoic acid (22:6 n-3) was present in PC and PE of liver, but was not detectable in peritoneal cell phospholipids. Peritoneal cells thus appeared to substitute longer chain n-6 PUFAs, namely, adrenic acid (22:4 n-6) and docosapentaenoic acid (22:5 n-6), for docosahexaenoic acid. A similar fatty acid pattern was also seen in the CE fraction of peritoneal cells, which was devoid of both docosahexaenoic acid and arachidonic acid but contained adrenic acid and docosapentaenoic acid. The enrichment of adrenic acid and docosapentaenoic acid in the CE and phospholipid lipid species of peritoneal cells (primarily macrophages) had
not been reported previously. Adrenic acid has been reported to be enriched in mouse kidney (27), human platelets (28), and rat pituitary (29, 30). Leukocytes have been described as possessing fatty acid elongase, but not fatty acid ⌬6 or ⌬5 desaturase activities (31– 34). Consequently, adrenic acid in peritoneal cells could be synthesized de novo by arachidonic acid elongation and/or obtained directly from the liver. In contrast, docosapentaenoic acid could not be synthesized de novo from arachidonic acid, but, instead, would most likely be obtained directly from the liver. The physiological significance of the enrichment of adrenic acid and docosapentaenoic acid in peritoneal cells is unknown. Both adrenic acid and docosapentaenoic acid could serve as a source of arachidonic acid via retroconversion (35). B. Lipid Remodeling Mediated by EFAD Comparison of the same tissues between mice fed an essential fatty acid sufficient or deficient diet revealed that there were general EFAD effects as well as tissuespecific EFAD effects. In general, EFAD led to the predominance of 16:0/18:1 and 18:1/18:1 as the major lipid species in both PC and PE of liver (Table 3; Figs. 3 and 4) and peritoneal cells (Figs. 3 and 4). These
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DUFFIN ET AL. TABLE 3
Quantification of Major Phosphatidylcholine and Phosphatidylethanolamine Lipid Species a Fatty acid 16:0
16:1 n-7
18:0
18:1 n-9
Total
A. Phosphatidylcholine species 18:2 n-6 species Corn oil EFAD 20:3 n-9 species Corn oil EFAD 20:4 n-6 species Corn oil EFAD 22:6 n-3 species Corn oil EFAD
2.7 ⫾ 0.1 0.14 ⫾ 0.01
nd b nd
1.3 ⫾ 0.0 nd
0.51 ⫾ 0.01 nd
4.5 0.14
nd 0.68 ⫾ 0.05
nd 0.06 ⫾ 0.01
nd 0.22 ⫾ 0.05
nd 0.30 ⫾ 0.07
nd 1.3
2.5 ⫾ 0.2 0.12 ⫾ 0.02
nd nd
1.9 ⫾ 0.2 0.30 ⫾ 0.07
0.97 ⫾ 0.08 0.10 ⫾ 0.04
5.4 0.52
1.6 ⫾ 0.0 0.05 ⫾ 0.01
nd nd
0.52 ⫾ 0.06 0.06 ⫾ 0.02
0.25 ⫾ 0.02 0.04 ⫾ 0.01
2.4 0.15
B. Phosphatidylethanolamine species 18:2 n-6 species Corn oil EFAD 20:3 n-9 species Corn oil EFAD 20:4 n-6 species Corn oil EFAD 22:6 n-3 species Corn oil EFAD
0.41 ⫾ 0.06 nd
nd b nd
0.62 ⫾ 0.06 nd
nd nd
1.2
nd 0.94 ⫾ 0.18
nd 0.10 ⫾ 0.01
nd 1.0 ⫾ 0.1
nd 1.8 ⫾ 0.1
nd 3.8
0.71 ⫾ 0.09 0.30 ⫾ 0.03
nd nd
2.3 ⫾ 0.3 0.35 ⫾ 0.03
1.9 ⫾ 0.3 0.69 ⫾ 0.09
4.9 1.3
3.8 ⫾ 0.4 0.58 ⫾ 0.03
nd nd
2.1 ⫾ 0.2 0.33 ⫾ 0.03
1.5 ⫾ 0.2 0.47 ⫾ 0.02
7.4 1.4
a A select subset of lipid species containing the key fatty acids, linoleic acid (18:2 n-6), Mead acid (20:3 n-9), arachidonic acid (20:4 n-6), and docosahexaenoic acid (22:6 n-3), was quantified in the livers of corn oil fed (i.e., essential fatty acid sufficient) and essential fatty acid deficient (EFAD) mice. The absolute amount of each phosphatidylcholine or phosphatidylethanolamine lipid species (g/mg of liver tissue) was determined by spiking a known amount of a nonbiological, phospholipid species into each sample as an internal standard. Results are expressed as mean ⫾ SE (n ⫽ 5 per group; two significant numbers). b Not detectable.
results clearly support past results showing induction and maintenance of ⌬9 desaturase activity consequential to the EFAD state (36). Another major difference in the fatty acid profile of liver and peritoneal cells from EFAD mice relates to the accumulation of Mead acid (20:3 n-9). In the liver, Mead acid was the major PUFA in all lipid classes, the major species being 16:0/20:3, 18:1/20:3, and 18:0/20:3 (Table 3; Figs. 3–5). In contrast, in peritoneal cells, there was no accumulation of Mead acid in CE and PE and only a trace amount in PC (Figs. 3– 6). These results showed the selectivity of Mead acid as an arachidonic acid surrogate in liver, but not in peritoneal, cells. The peritoneal cells, then, became depleted of arachidonic acid upon the advent of essential fatty acid deficiency, yet Mead acid did not substitute for arachidonic acid. The only major change in the peritoneal cells was the dramatic increase in monoenoic lipid species, 16:0/18:1, 18:1/18:1, and 16:1/ 16:1, in PC and PE (Figs. 3 and 4). Finally, the EFAD
FIG. 6. Composition of cholesterol ester species in the livers of essential fatty acid sufficient (i.e., corn oil fed) and essential fatty acid deficient mice.
TANDEM MASS SPECTROSCOPIC ANALYSIS OF LIPID REMODELING
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