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Anal. Chem. 1985, 57,710-715
Carbon- 13 Nuclear Magnetic Resonance Spectrometric and Gas Chromatography/Mass Spectrometric Characterization of Lipids in Corn Suspension Cells Dennis James Ashworth,* Douglas Owen Adams, Benjamin Yunwen Giang, Michael Tung-hai Cheng, a n d R h o Yul Lee Western Research Center, Stauffer Chemical Company, 1200 South 47th Street, Richmond, California 94804
Carbon-13 NMR spectrometry has been applled in conjunctlon wlth gas chromatography/mass spectrometry to the characterization of llpld dlstrlbutlon and structure in suspenslon cells of corn (Zea mays var. Black Mexican Sweet). Growth of the cells In the presence of sodium [l-13C]acetate or [2-13C]acetate followed by NMR analysls of the extracted llplds displayed an lnltlal lncorporatlon of acetate Into oleate (18:l). After 24 h, NMR analysis suggested that >90% of the labeled oleate had been converted to llnoleate (18:2). Integratlon of the enrlched carbonyl resonances of the neutral and polar Ilpld's fatty aclds showed an even dlstrlbutlon of the label between the two lipid groups. 31PNMR spectrometry showed the polar llplds to be primarily represented by phosphatldylcholine. GC/MS analysls of the derlvltlzed fatty aclds suggested that the oleate/llnolenate ratlos may be characterlstlc of the respectlve netural and polar lipids.
Table I. Growth Inhibition of Nonphotosynthetic Corn Suspension Cells (Zea mays var. Black Mexican Sweet) by Sodium, Potassium, and Ammonium Acetatea concn, mM
sodium acetate
potassium acetate
ammonium acetate
0 0.5
100.0 99.1
100.0 86.6 92.1 87.5 67.9 46.6 32.7
100.0 91.3 102.9 95.3 62.1 14.6 32.1
1.o
100.0
2.5 5.0
83.7
7.5 10.0
49.0 30.0
93.3
a Cultures were grown 14 days at the indicated acetate concentration (in triplicate) and the final packed cell volumes determined. Results (average) are expressed as percentage of control packed cell volume.
of the fatty acids and the characterization of the structure of the intact lipids. In the course of investigating the effects of various herbicides on fatty acid biosynthesis in suspension cells of corn, a necessary prerequisite was the characterization of the lipid components prior to herbicide treatment (1). In plant cells, the various polar and neutral lipids are generally composed of C16(palmitic, 16:O; palmitoleic, 161) and C18 (stearic, 18:O; oleic, 18:l; linoleic, 18:2 linolenic, 18:3) saturated and unsaturated fatty acids esterified to the 1 and 2 positions of a glycerol backbone. Modifications to the 3 position of glycerol by the addition of monoalkyl phosphates, sugars, or additional acyl groups then generates the three major classes of lipids (2).
The biosynthesis of C16 and Cls fatty acids consists of the acyl carrier protein (ACP) mediated head to tail linkage of consecutive acetate units to generate palmitoyl-ACP (3-6). Further elongation of this substrate by the transfer of a two-carbon unit from malonyl-ACP yields stearoyl-ACP which undergoes an initial unsaturation to generate oleayl-ACP (7). Further details as to the transitional events which lead from the fatty acid-ACP complex through free acids and/or coenzyme A esters to neutral and phospholipid formation are less clear (8). Likewise, the specific metabolic steps leading to the more highly unsaturated linoleic (18:2) and linolenic (18:3) fatty acids have yet to be firmly established (9-12). Carbon-13 nuclear magnetic resonance (NMR) spectrometry has emerged as an extremely powerful tool for structural analysis of biomolecules and the observation of metabolic processes (13-18). In the studies reported here, the metabolism of carbon-13 labeled acetate to various saturated and unsaturated fatty acids in suspension cells of nonphotosynthetic corn has been monitored by NMR spectrometry. In addition, gas chromatographic/mass spectrometric (GC/MS) characterization of the lipid derived fatty acid methyl esters has been performed to aid in the analysis of the distribution
MATERIALS A N D METHODS Suspension cells of corn (Zea mays var. Black Mexican Sweet) were used in all experiments and were cultured in a modified (2 mg/L 2,4-D and 132 mg/L L-asparagine) Linsmaier and Skoog medium (19). The nonphotosynthetic cells were transferred weekly under aseptic conditions with 15 mL of 7 day old cells being transferred into 25 mL of fresh medium. One milliliter of 7 day old culture contained approximately 150 mg of fresh weight of cells. All culture flasks were maintained at 27 OC under conditions of 16 h of light and 8 h of dark on a rotary shaker at 125 rpm. A preliminary experiment was conducted to establish the maximum concentration of acetate which could be used in the metabolism study without inhibiting cell growth and to evaluate the effect of various acetate cations (Na', K+, and NH,+) on growth. Flasks containing 10 mL of fresh medium were inoculated with 2 mL of 7 day old suspension cells. To each respective culture (in triplicate) was then added sodium, potasium, or ammonium acetate to give the concentrations shown in Table I. The cells were grown 14 days at 27 "C and the final packed cell volumes were determined by pouring the contents of each culture flask into a graduated 15-mL centrifuge tube and centrifuging at 550g for 2 min. The starting packed cell volume of each flask prior to acetate addition was approximately 0.8 mL. Preparation of corn suspension cells prior to NMR analysis was initiated by transferring 15 mL of 7 day old cells into 25 mL of fresh medium. The cells were given 3 days to stabilize in the new culture medium at which time sodium [l-13C]acetateor [2-13C]acetate was added to a final concentration of 2.0 mM (except where indicated). The cells were then harvested after a specific time following acetate addition. One flask of cells was used for each time point. Cells were collected by vacuum (aspirator) filtration on to Whatman No. 1 filter circles (9 cm) for 1 min with concurrent distilled water washing to remove any extracellular acetate or medium. The fresh weight of the cells was then determined. A typical 40-mL culture contained 3.4 g fresh weight of cells at 2 h. The isolated cells were extracted according to Bligh and Dyer (20) to separate water and chloroform soluble metabolites. Following concentration of the chloroform (lipid) fraction,
0003-2700/85/0357-0710$01.50/00 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 3,MARCH 1985
the residue was dissolved in 0.6 mL of CDC1, for NMR analysis. Carbon-13 labeled sodium [l-13C]acetate (99%) and sodium [213C]acetate(99%) were purchased from MSD Isotopes and were used without further purification. Fractionation of the lipids into polar and neutral classes was performed by dissolving the chloroform residue (approximately 25 mg) from the Bligh and Dyer extraction in 10 mL of diethyl ether. One gram of activated silicic acid was added and the suspension was allowed to stir at 5 "C for 30 min. After centrifugation the resulting ether layer was collected and the silica was washed two additional times with 10 mL of ether followed by centrifugation and ether collection. Concentration of the combined ether extract yielded the separated carbon-13 enriched neutral lipids for NMR and GC/MS analysis. The phospholipids were recovered by washing the silica with 3-10 mL portions of methanol followed by centrifugation, methanol collection, and concentration (21). Base hydrolysis of isolated lipids and subsequent fatty acid esterification was performed in 10 mL of 2 N NaOH for 2 h at 100 "C. The cooled solution was acidified with 2 N HC1 and the free fatty acids were extracted with 2-10 mL portions of diethyl ether. Concentration and addition of ethereal diazomethane to the residue generated the correspondingmethyl esters. Acid catalyzed transesterification of the lipids was performed by the addition of 10 mL of methanol, containing 0.1 mL of trifluoromethanesulfonic acid, to the lipid residue. Refluxing at 65 "C for 2 h followed by concentration of the solution yielded the methyl esters. Carbon-13 NMR spectrometry was performed on a Varian XL-200 NMR spectrometer operating at 50.1 MHz. All spectra were obtained at 28 "C employing an 11kHz spectral width (1.45 s acquisition time) and 8.8 ps pulse width (50" pulse angle) with continuous broad-band 'H decoupling for 32K data points. Spectra were acquired for 38K transients and resonance chemical shifts are relative to deuteriochloroform (77.0 ppm). Phosphorus-31 NMR spectra were recorded on the same instrument at 81.0 MHz utilizing the same spectral width, pulse width, and data points as in the carbon-13 NMR experiments. Phosphorus-31 chemical shifts are relative to 85% H3P04(0.0 ppm). GC/MS analysis was performed on a Finnigan-MAT Model 4021 gas chromatograph/mass spectrometersystem equipped with a Model 9600 gas chromatograph. Fatty acid esters were separated on both a 1.8 m 10% OV-101 packed column and a 30 m X 0.25 mm (i.d.) DB 1701 fused silica capillary column with injection port temperatures of 210 "C and helium carrier gas. Both column oven temperatures were programmed from 40 "C to 200 "C at 30 "C/min and then from 200 "C to 250 "C at 4 "C min. Samples were dissolved in dichloromethane to a concentration of 0.5-2.0 pg/pL and 1.0 pL was inejected (501 split). The electron impact mass spectra were obtained at 70 eV electron energy and 250 pA emission current.
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NATURAL ABUNDANCE
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Flgure 1. Carbon-13 NMR spectra of isolated corn cell lipids following culture growth for 7 h in (A) normal medium, (B) medium and 2.0 mM sodium [2-%]acetate, and (C) medium and 2.0 mM sodium [ l '3C]acetate. Resonance assignments correspond to major labeled fatty acids present: P, palmitate; 0, oleate; L, linoleate.
NATURAL ABUNDANCE
C
RESULTS Initial experiments were performed to obtain the natural abundance carbon-13 NMR spectra of the total extracted corn cell lipids. Extraction of the lipids prior to acetate treatment and extraction following 2.0 m M sodium [1-l3C]acetate and 2.0 mM [2-13C]acetatetreatment for 7 h produced the three respective spectra shown in Figure 1. The natural abundance carbon-13 spectrum (Figure 1A) allowed observation of all extracted lipid carbons. However, because of the large number of resonances present, with considerable overlapping of resonances in many cases, the assignment of specific resonances to particular fatty acids was difficult. This difficulty was reduced, as was an increase in sensitivity gained, when the suspension cells were treated with 2.0 m M sodium [2J3C]acetate (Figure 1B) or sodium [l-13C]acetate(Figure 1C) for 7 h prior to lipid extraction. A comparison of the resonance chemical shifts observed in Figure 1 parts B and C t o thhe carbon chemical shifts obtained from the appropriate authentic reference compounds (Table 11),allowed the tentative identification of oleic and linoleic acids as constituents of the isolated lipids since the route of fatty acid biosynthesis from 1- or %labeled acetate results in fatty acids with alternate carbons labeled (22). All remaining resonances in spectra A,
133
131
128
126ppm
Figure 2. Olefinic region expansion of extracted lipids carbon-13 NMR spectra obtaned from nonphotosynthetic corn suspensions cells grown for 7 h in (A) normal medium, (B) 2.0 mM sodium [2-%]acetate, and (C) 2.0 mM sodium [ l-'3C]acetate: 0, oleate; L, linoleate.
B, and C could not be unambiguously assigned to specific fatty acids due to the lack of unique carbon chemical shifts for the remaining aliphatic carbons of palmitic, palmitoleic (22), stearic (23), oleic, linoleic, and linolenic acids. Further characterization of the carbon-13 enriched lipid components was obtained by examination of the olefinic regions of the respective spectra (Figure 2). All spectra (Figure 2A-C) lack resonances assignable to linolenic acid (130.1,127.7, 128.2, 128.1, 127.0, and 131.8 ppm) with the 131.8 and 127.0 ppm resonances being the most distinct resonances absent in the spectra. However, spectra in Figure 2 parts B and C do allow the identification of oleic and linoleic acids as major enriched lipid components due to each respective fatty acid's unique olefinic carbons chemical shifts. These results
712
ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985
713
LIPID MIXTURE A
B
4
6
E
I
175
131
I
129
127ppm
Flgure 3. Carbon-I3 NMR spectra of [2-13C]acetateincorporation into unsaturated fatty acids of corn suspension cells. Cultures were grown under identical conditions for the indicated length of time followed by
extraction of the lipids from the cells. All samples were dissolved in 0.6 mL of CDCI, and 13C spectra were acquired under Identical conditions. Spectra are displayed in the absolute intensity mode: 0, oleate; L, linoleate.
rl
I\ n I
173
NEUTRAL LIPIDS
I 171 ppm
Figure 4. Carbonyl region expansion of 13C NMR spectra of (A) lipid mixture obtained from extracted corn suspension cells, (B) separated
neutral lipids, and (C) polar lipids obtained from lipid mixture. Cells were grown 8 h in the presence of 2.0 mM sodium [ l-13C]acetateprior to lipid extraction.
resonance corresponding to the Clo of oleate. The appearance of the Clo resonance of oleate after just 2 h suggests some carbon-13 enrichment from acetate already at this position since the Cg of oleate would also be observable just upfield of the C9 of linoleate at 129.6 ppm if the oleate Clo was not therefore supported the identification of oleate and linoleate already enriched. Because treatment of the cells with [2obtained from analysis of the aliphatic region (Figure 1). 13C]acetateenriches only the even-numbered carbons of oleate The identification of palmititic acid as a major lipid comand linoleate, after 4 h (Figure 3B) the Clo position of oleate ponent of these plant cells was accomplished due to methyl becomes further enriched. The Cg of linoleate, not enriched palmitate's early, well-resolved gas chromatographic retention by [2-13C]acetate,is now masked by the oleate Clo while the time. However, in the course of standardizing hydrolysis and Clo and C12of linoleate are still at natural abundance intensity. methylation conditions for GC/MS identification of the lipid After 6 h (Figure 3C), all even-numbered olefinic carbons were derived esters, an authentic sample of 1-palmitoyl-2further enriched until a maximum was reached at 8 h (Figure linoleoyl-~-glycero-3-phosphorylcholine was used. Upon acid 3D) a t which time no extracellular [2-13C]acetateremained in the medium (depleted a t approximately 7 h). After 24 h F1 c H,- o -C - (C H , ) , ~ CH, however, it can be observed that all labeled oleate had been I 0 il depleted, yet the incorporation of label into linoleate had -~-C-(CH,),-CH=CH-CH,-CH=CH-(CH~)~-CH, continued. These observations suggest a flow of carbon from I F 1 Y 3 acetate, through oleate to linoleate. The same results were CH2-O-~-O-CHz-CH2-N-CH3 observed by the repeat of the experiment using [l-13C]acetate. 0c H3 Further characterization of the lipids was accomplished by l-palmitoyl-2-linoleoyl-~-glycero-3-phosphorylcholine separating the mixture into polar and lipid fractions. While catalyzed methanolysis in dilute trifluoromethanesdfonic acid, carbon-13 NMR analysis of the olefinic and aliphatic regions only methyl palmitate was observed in the GC/MS analysis of the separated components only displayed, in the neutral of the sample. Yet when 2 N aqueous sodium hydroxide was fraction, an approximate 15% increase in oleate relative to used for lipid hydrolysis followed by ethereal diazomethane linoleate, an examination of the enriched ester carbonyl regions esterification, both methyl palmitate and methyl linoleate were did reveal distinctive carbonyl carbon chemical shifts for the observed by GC/MS. This result suggested that under the respective polar and neutral fractions and for the specific 1acidic conditions employed for lipid methanolysis, only fatty and 2-acyl chains (Figure 4). This observation is consistent acids esterified to the 1and 3 positions of glycerol were being with the chemical shifts reported in Table I1 and has been cleaved possibly due to steric hinderance of the methanol explained by steric interactions with the glycerol carbons and approach to the 2-acyl linkage (aqueous 2 N HCl did cleave adjacent acyl chains (23-26). The distinct carbonyl chemical the 2-acyl linkage). This same terminal position specificity shifts observed in the separated polar and neutral lipids was also observed when l-oleoyl-2-palmitoyl-~-glycero-3- (Figure 4B, C) suggest that integration of the most upfield phosphorylcholine was treated with dilute methanolic tri(neutral lipid 2-acyl carbonyl) and most downfield (polar lipid fluoromethanesulfonic acid. This observation was used to 2-acyl carbonyl) carbonyl resonances of the total extracted further characterize the lipid structures. lipid mixture (Figure 4A) could qualitatively allow the cellular Carbon-13 NMR analysis of the time course of [2J3C]polar/neutral lipid ratio to be determined. acetate incorporation into lipids is shown in Figure 3. ObPhosphorus-31 NMR analysis of the separated polar lipids served in the olefinic expansion (Figure 3A) are essentially qualitatively showed phosphatidylcholine to be the major the natural abundance signals of linoleate and one additional phophorus-containing component with resonances corre-
th
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985
cultured cells following 1 h of acetate treatment to the fatty acid distribution obtained from a culture grown 24 h in 2.0 mM acetate showed that acetate feeding did not alter the fatty acid distributions.
PC
DISCUSSION
PS
I
I
\
I
2
\ I
I
0
-bpm
Flgure 5. Phosphorus-31 NMR spectrum of extracted polar lipids from
corn suspension cells. Resonance assignments are based on comparisons of chemical shifts to authentic reference compounds: phosphatidylcholine (PC), -0.4 ppm; phosphatidylinosital, -0.1 ppm (broad);phosphatidylethanolamine(PE), 0.5 ppm; phosphatidylserine (PS), 0.9 ppm (broad):and phosphatidylglycerol,1.8 ppm. Chemical shifts are relative to external 85% H,PO, (0.0 ppm). sponding to phosphatidylethanolamine and phosphatidylserine also present (Figure 5). The ratio of these resonances did not change throughout the time course (2-24 h) experiments. Characterization of the total (carbon-13 labeled and unlabeled) fatty acid pools was achieved by hydrolysis and methylation of the lipids to the corresponding fatty acid methyl esters (FAMES) followed by GC/MS analysis. The distribution of FAMES obtained by base hydrolysis of the separated neutral and polar lipid fractions is shown in Table 111,samples A and B. A considerable difference can be observed between the oleate and linolenate distributions. The neutral lipids contain approximately two times the oleate yet only one-fourth the linolenate as compared to the polar lipid sample. This difference was also observed upon acid catalyzed methanolysis of the neutral and polar lipid fractions (Table 111, samples C and D). In this case three times more oleate and one-seventh the linolenate were found in the neutral lipids as compared to the polar. Because of the selective l-acyl transesterification with trifluoromethanesulfonic acid methanolysis, a comparison of the later oleate and linolenate ratios (Table 111,samples C and D) to the former (samples A and B) oleate and linolenate ratios suggest a fairly uniform distribution of the observed fatty acids among the 1, 2, and 3 positions of the triacylglycerols and the 1and 2 positions of the remaining lipids. Clearly linoleate appears to occupy approximately half of all lipid fatty acid positions in the separated polar lipids (Table 111, sample B) and nearly two-thirds of the fatty acid positions in the neutral lipids (Table 111,sample A). This was observed whether the free FAMES were generated by acid catalyzed methanolysis or base hydrolysis. Therefore the linoleate as well as the other observed fatty acids appear to be evenly distributed. A comparison of the fatty acid distributions when a mixture of polar and neutral lipids were hydrolyzed in base to the distribution when hydrolysis/ methanolysis was catalyzed by acid (selective 1-and 3-acyl hydrolysis) showed a preferential 1-and/or 3-position esterification of linoleate to the glycerol backbone and a preferential %position enrichment with oleate (Table 111,samples E and F). This result is substantiated by a comparison of samples A and B and samples C and D in Table 111. Finally, a comparison of the distribution of fatty acids obtained from the
The results presented here describe the use of NMR spectrometry for the observation of in vivo synthesis of fatty acids from acetate in extracts of corn suspension cells and the structural features of specific fatty acids and intact lipids which can be obtained by the use of NMR spectrometry in conjunction with gas chromatography/mass spectrometry. The results obtained from the observation of [ l-'3C]acetate and [2J3C]acetate incorporation into fatty acids as a function of time (Figure 3) suggest a flow of the labeled carbons into oleate by way of palmitate and stearate, and finally into linoleate (27). GC/MS analysis of the total labeled and unlabeled fatty acid pool showed significant (approximately 10% of total) linolenate to be present in the cells. However, incorporation of carbon-13 labeled acetate into linolenate was not observed (28) and continued culture growth up to 72 h likewise did not show detectable conversion of the labeled oleate or linoleate to linolenate. Whether linolenate is derived from desaturation of linoleic acid ( I I ) , linoleoylphosphatidylcholine (29),linoleoylmonogalactosyldiglyceride (121, or from elongation of dodecatrienoic acid (10) is yet to be firmly established. Nevertheless, the results obtained here fail to show the conversion of carbon-13 labeled linoleate to linolenate after 24 h of growth in 2 mM sodium acetate. The rapid increase in oleate labeling followed by its subsequent near disappearance and the concurrent increase in linoleate labeling has been observed by using other methods of analysis (11,12,27,30,31). The result suggests a relatively small rapidly turning-over pool of oleate since >go% of the labeled oleate observed following 8 h of cell growth in 2 mM [2-13C]acetate had disappeared by 24 h. GC/MS analysis (Table 111) also sugests a relatively small pool of oleate. The results obseved here also suggest that desaturation of oleate to linoleate as well as elongation of palmitate to oleate, via stearate, occurs while the respective fatty acids are esterified (32). This conclusion is based on carbon-13 NMR analysis which failed to show the presence of any detectable free labeled fatty acids or carboxylates in the spectra of the isolated lipids. While NMR analysis enabled observation of the flow of labeled acetate into lipids and identification of the unsaturated fatty acid esters, mass spectrometric analysis allowed identification of unresolved (in the NMR spectra) fatty acids and the determination of the individual fatty acid pool sizes. The results show linoleate to be the most abundant fatty acid present in the polar and neutral lipids occupying over half the acyl sites in the isolated lipids. Palmitate was found to be the next most abundent fatty acid occupying approximately 20% of the remaining polar and neutral lipid acyl positions. Interestingly, the presence of palmitoleate (16:l) was not observed. This is consistent with other findings suggesting localization of palmitoleate in photosynthetic tissue (2, 11, 27, 31, 33). Stearate was found to be present in low (approximately 1% of total) yet constant abundance and was evenly distributed between polar and neutral lipid fractions. Perhaps most interesting were the reciprocal distributions of oleate and linolenate between the polar and neutral lipids. When oleate comprised a relatively high percent (10-12%) of the total lipid in the separated neutral lipids, linolenate was found to be relatively low (approximately 3%). Conversely, when linolenate was relatively high (17-22%) in the polar lipids, oleate was found to be low (3-5%). The uniform distribution of palmitate, stearate, and linoleate observed in both polar and neutral lipid fractions suggest that the high
ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985
oleate-low linolenate in neutral, high linolenate-low oleate in polar lipids may be characteristic of the lipid structures in nonphotosynthetic corn suspension cells.
CONCLUSIONS The picture that emerges from the work on corn suspension cells presented here is one of neutral and polar lipids composed of palmitic, stearic, oleic, linoleic, and linolenic acids. Growth of the cells in [l-13C]acetateand [2-13C]acetatequickly enriches palmitate and oleate. The labeled oleate is then converted into linoleate with little (
