J Am Oil Chem Soc (2013) 90:1495–1502 DOI 10.1007/s11746-013-2296-y
ORIGINAL PAPER
Fatty Acid Composition in Ergosteryl Esters and Triglycerides from the Fungus Ganoderma lucidum Zi-Long Deng • Jian-Ping Yuan • Yan Zhang Xiao-Ming Xu • Chou-Fei Wu • Juan Peng • Jiang-Hai Wang
•
Received: 22 March 2013 / Revised: 27 April 2013 / Accepted: 22 June 2013 / Published online: 10 July 2013 Ó AOCS 2013
Abstract Free and esterified ergosterols are detected almost solely in fungi and are often employed as a biomarker of living fungi. In this work, the fatty acid composition and d13C values of major fatty acids in triglycerides and ergosteryl esters from the fungus Ganoderma lucidum were analyzed by gas chromatography– mass spectrometer and gas chromatography–isotopic ratio mass spectrometer, respectively. The results showed that the fatty acid profiles varied in triglycerides and ergosteryl esters. The percentage of saturated fatty acids in ergosteryl esters was remarkably higher than that in triglycerides, where CD9c 18:1 was the predominant fatty acid and constituted 61.26 % of the total fatty acids. In contrast, C16:0 was the predominant fatty acid and constituted 71.88 % of the total fatty acids in ergosteryl esters. The study suggests that, after fungal death, free ergosterols in the cell membrane of the dead fungus were esterified with preferentially saturated fatty acids, mainly C16:0, from triglycerides and then stored in lipid particles for a longer period while free ergosterol markedly decreased. The d13C values of C16:0, C18:0, C18:1 and C18:2 in ergosteryl esters exhibit a
Z.-L. Deng and J.-P. Yuan contributed equally to this work. Z.-L. Deng Y. Zhang X.-M. Xu C.-F. Wu J.-H. Wang (&) Guangdong Provincial Education Department Key Laboratory of Marine Petroleum Exploration and Development, School of Marine Sciences, Sun Yat-Sen University, Guangzhou 510006, People’s Republic of China e-mail:
[email protected] J.-P. Yuan (&) J. Peng Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-Sen University, Guangzhou 510006, People’s Republic of China e-mail:
[email protected]
pronounced depletion in 13C compared with that in triglycerides within the range of -1.3 to -0.9 %, supporting the above inference. It is again suggested that free ergosterol in the cell membrane should be used as an indicator of living fungi, and ergosteryl esters in the lipid particles should not be included in the measurement of living fungal biomass. Keywords Fatty acids Ergosteryl esters Free ergosterol Stable carbon isotopes Gas chromatography–mass spectrometer (GC–MS) Gas chromatography–isotopic ratio mass spectrometer (GC–IRMS) Ganoderma lucidum
Introduction Sterols, synthesized in the endoplasmic reticulum but enriched in the plasma membrane, represent a crucial class of lipids and are indispensable constituents of all eukaryotic cells [1, 2]. Therefore, the sterols as the constituents of membranes may make an impact on various membranerelated functions, such as maintaining the permeability and fluidity of the cell membrane [1, 3]. In order to preclude the harmful accumulation of free sterols synthesized in the endoplasmic reticulum, the free sterol has to be efficaciously transferred to and enriched in the plasma membrane [1, 2]. In addition, an excess of free sterols also esterified with fatty acids to steryl esters as a storage form of sterols [1]. The relatively more abundant esters are formed when the fungus is going into a stationary phase [4]. Therefore, ergosterol in fungi occurs in two forms, free and esterified ergosterols [5, 6]. The esterification of free ergosterol and the turnover of ergosteryl esters play a key part in preserving ergosterol
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homoeostasis, which includes sterol biosynthesis, esterification for storage and turnover for utilization [7]. The hydrolysis of ergosteryl esters is as important as the esterification of ergosterol for sterol homeostasis and a flexible ergosteryl ester pool may give the benefit of preventing the cell from both an excess and deficiency of free ergosterol [3]. Upon requirement, ergosteryl esters are hydrolysed and free ergosterol is used as a building block for the construction of cell membranes in fungi [7]. In fungi, the synthesis, storage and hydrolysis of ergosteryl esters are processes related to lipid storage [3]. Neutral lipids including triglycerides and ergosteryl esters are important storage molecules as stored energy and source of building blocks for the construction of cell membranes [3, 7]. Because of being unable to be incorporated into phospholipid bilayers, triglycerides and ergosteryl esters gather and grow into the hydrophobic core of a lipid particle [3]. Ergosterols are found almost solely in fungi and are often used as a biomarker of living fungi based on the supposition that ergosterols are unstable and thus quickly decompose after fungal death. Separation of ergosterols into free and esterified forms might give some additional information on the vitality of the fungal mycelium [4]. It was reported that the hymenophores and the spores had a substantially higher proportion of ergosteryl esters than the pilei and the stipes in the fungus Ganoderma lucidum [5]. The previous study also showed that the ergosteryl esters were more steady than free ergosterol, and even an increase in the esterified ergosterol level was also detected in pulverizing dead fungal hyphae [6], suggesting that free ergosterol might be esterified and stored in lipid particles. Therefore, it has been suggested that free ergosterol in fungal cell membrane should be used as a biomarker of living fungi and ergosteryl esters in the lipid particles should not be included in the measurement of living fungal biomass [5, 6]. Although it was reported that the prevailing fatty acids in ergosteryl esters were C16 and C18 unsaturated fatty acids in yeast [8], little was known of the prevalent fatty acids in ergosteryl esters and triglycerides in G. lucidum. In the present study, the fatty acid compositions and stable carbon isotope ratios of the dominant fatty acids in ergosteryl esters and triglycerides from G. lucidum were studied.
Materials and Methods Reagents and Treatment The solvents and reagents, including sulfuric acid, acetic acid, tetrahydrofuran (THF), ethyl acetate, petroleum ether
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(PE) (its boiling point, 30–60 °C), ethyl ether, dichloromethane (CH2Cl2) and anhydrous sodium sulfate, were all of analytical grade and were provided by Yueshen (Guangzhou, China). PE was purified by rinsing three times with concentrated sulfuric acid followed by a 2 % Na2CO3 solution and water, drying over anhydrous sodium sulfate and distilling with a rectifying column. Dichloromethane, ethyl acetate and ethyl ether were purified by distilling with a rectifying column. Methanol [Merck, high-performance liquid chromatography (HPLC) grade], THF and anhydrous PE were obtained by refluxing the solvents with sodium wire followed by redistillation, and the d13C values of anhydrous methanol was detected in an individual gas chromatography–isotopic ratio mass spectrometer (GC–IRMS) run. Sodium methoxide (5 %) was prepared by adding the estimated amount of fresh sodium wire to anhydrous methanol. Sample Preparation The fruiting bodies of G. lucidum for this study were sampled from the log-cultivated Ganoderma base in Fujian, southeast China. The fruiting body sample was desiccated at 50 °C before being manually milled with a grinder into a 60–120 mesh powder. The dried sample of 100.03 g (\120 mesh) was Soxhlet-extracted with CH2Cl2/ methanol (8:1, v/v) for 72 h. The suspension was filtered, and the solvent was removed with a rotary evaporator at 40 °C and a flow of nitrogen. The total extract of 3.33 g and PE-soluble extract of 2.10 g were accordingly obtained on a dry weight basis. Separation and Purification of Ergosteryl Esters The column chromatograph (CC) method for the separation and purification of PE-soluble composition was established by optimizing the separation conditions using the thin-layer chromatograph (TLC) method. The result indicated that the best ratio of the initial eluant was 20 % CH2Cl2 to 80 % PE. A preparative column was filled with activated silica gel. The inner diameter of the column was 13 mm, and the height of the silica gel was 100 mm. Twenty percent dichloromethane and 80 % PE were used to elute the mixture. During the separation, the nitrogen pressure was maintained to control the elution speed of 2.5 mL/min. The column capacity was measured in this way and every 3 mL of the elution reagent was gathered into one tube. During the purification, the TLC and HPLC methods were adopted to monitor the process of chromatographic separation.
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Analysis of Ergosteryl Esters Ten milligrams of ergosteryl esters was dissolved in 0.8 mL of toluene in a test tube, and sodium methoxide in a 2 mL anhydrous methanol solution was added before the mixture was kept 3 h in a stoppered tube at 70 °C. Hydrochloric acid was added to the mixture and kept for 10 h at 80 °C. The PE layer was rinsed three times with pure water. The PE layer including fatty acid methyl esters (FAME) was dehydrated with anhydrous sodium sulfate and filtrated. One milliliter of freshly fabricated anhydrous THF and 1 mL of 5 % sodium methoxide were added to a solution of triglycerides purified from G. lucidum. Fifty milligrams of triglycerides were added to 2 mL of anhydrous PE in a penetrable screw capped vial with a dry syringe. After agitating and standing for 10 min, the mixture was neutralized with 1 mL of 5 % acetic acid and washed three times with pure water. The PE layer including FAME derived from triglycerides was desiccated with anhydrous sodium sulfate and filtrated. Ten milligrams of ergosteryl esters was dissolved in 0.8 mL of toluene in a test tube, and 1 % sulfuric acid in 2 mL of methanol was added before the mixture was kept overnight (or reflux 5 h) in a stoppered tube at 80 °C. Two milliliters of water containing 5 % sodium chloride was added and the required esters were extracted with hexane (2 9 5 mL), using Pasteur pipettes to separate the layers. The hexane layer was washed with 4 mL of water containing 2 % potassium bicarbonate and desiccated over anhydrous sodium sulfate. The solution was filtered and the solvent was removed under a stream of nitrogen. The base hydrolysis of ergosteryl esters derived from G. lucidum was monitored by using the TLC method. The result demonstrated that the Rf value was 0.48 in the mobile phase consisting of 95 % PE and 5 % ethyl acetate, implying that the base hydrolysis was completed. Transesterification One milliliter of freshly fabricated anhydrous THF and 1 mL of 5 % sodium methoxide were added to a solution of about 50 mg samples of ergosteryl esters and triglycerides in 2 mL anhydrous PE in a passable screw-capped vial with an dry syringe. After agitating and standing for 10 min, the mixture was neutralized with 1 mL of 5 % acetic acid and washed three times with pure water. The FAME-bearing PE layer was dehydrated with anhydrous sodium sulfate and filtered. Analysis of Individual FAME Individual FAME were saponified with a sodium methoxide (5 %) solution in 95 % methanol for 3 h at 70 °C.
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Then, the reaction mixture was neutralized and extracted with ethyl ether, and the individual fatty acid was attained by evaporation of the solvent with a stream of nitrogen. The FAME samples in PE were analyzed on the Agilent Technologies 6890 gas chromatograph (Agilent, Palo Alto, USA) fitted out with a Gerstel MPS2 autosampler (Mu¨lheim an der Ruhr, Germany), a Varian fused silica column (CP-7419, Palo Alto, USA), and an Agilent 5973 mass spectrometer (Palo Alto, USA). The experimental conditions were adopted as follows: helium was the carrier gas at a fixed flow rate of 1.0 mL/min; the split proportion was 10:1 for injection in the split mode; the initial oven temperature was 100 °C and held for 2 min; the first program rate was 6 °C/min to 190 °C and held for 5 min; the second program rate was 20 °C/min to 280 °C and held for 8 min; the voltage of its electron ionization was 70 eV; the temperature of its source was 250 °C; and its mass scan values were within the range of 40–600. Before sampling, a blank run was carried out to assure no carryover of analytes from previous extraction. The samples were detected three times and the standard deviations of their relative amounts were all less than 0.3 %. GC–IRMS The d13C values of individual FAME were measured on an HP 6890 GC (Hewlett-Packard, Palo Alto, USA) fitted out with a split/splitless injector integrated via a combustion oven to an IsoPrime IRMS (Manchester, UK). The combustion interface included copper wires, which were doped with oxygen (CuO) and sustained at 960 °C. Samples were sampled into the GC–IRMS in a splitless mode by a helium gas stream at a fixed flow rate of 1.0 mL/ min. The injector temperature was sustained at 290 °C. The compound separation was carried out on an identical column in accordance with the aforesaid temperature program. The relative contents of carbon isotopes were detected by using the mass spectrometer. For the calculation purpose, the reference gas of carbon dioxide was simultaneously pumped into the IRMS in multiple pulses at the initial and final procedures for each analysis. This permitted the detection of the d13C value for the sample in comparison with that of the reference gas of carbon dioxide. Furthermore, a standard mixture including ten n-alkanes with the foregone isotopic composition was measured two or three times per day to examine the instrument situation, and the chromatogram peak of a tested compound should fall in a half to double of the reference gas peak, or the tested value should be considered to be inaccurate beyond the detection limit. The carbon isotope composition is expressed by the conventional d-notation (%): d13 CPDB ð&Þ ¼ ½ðRsample =Rstd Þ 1 103
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ð1Þ
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where Rsample and Rstd represent the 13C/12C isotope ratios of the sample and international V-PDB carbonate standard, respectively [9]. The total analytical error (i.e., standard deviation) for this method was superior to 0.1 %. Additionally, a standard mixture including ten n-alkanes with its foregone isotopic composition was measured twice daily to examine the precision and reproducibility. The measured deviation from the real value of the n-alkanes standard was commonly less than ±0.5 %. Three parallel analyses were simultaneously conducted on our samples. Their standard deviations were in the range of 0.2–0.3 %, and exhibited the excellent reproducibility. A correction of the d13C value for each FAME was necessary, because each FAME included a methyl group derived from trans-esterification that did not occur in the native lipid. The carbon isotopic ratio of the methyl group was analyzed by detecting the d13C value of the methanol reagent adopted to methylate the acids. The d13C mean of the methyl group in sextuplicate was -32.5 ± 0.2 %. With a foregoing carbon isotopic ratio of the methyl group, the original d13C values of the major fatty acids in samples were corrected as follows: d13 CFA ¼ ½ðn þ 1Þ d13 CFAME d13 CMethyl group =n
ð2Þ
where FA and n represent fatty acids and the carbon atom numbers of fatty acids, respectively [10]. The standard deviations of the corrected d13C values of the primordial fatty acids ranged from 0.2 to 0.3 %. Elemental Analysis–Isotopic Ratio Mass Spectrometer (EA–IRMS) To detect the carbon isotopic ratios of the fruiting body of G. lucidum and its PE-soluble extract, ergosteryl esters and triglycerides, nearly two grams of each sample were weighed and wrapped in a clean Sn capsule. The capsulated samples were located in a CE EA1112 elemental analyzer (CE Instruments, UK), and combusted in an oxygen atmosphere in a burning CuO tube at 960 °C. Combustion gases were washed out via a reduction column by a helium gas stream and entered the gas chromatograph where carbon dioxide, still in the helium stream, was isolated from the other gases. The gas stream then passed into a Delta plus mass spectrometer (Finnigan, USA), where the carbon dioxide gas was measured in comparison with the carbon dioxide reference gas of a foregone d13C value (-29.5 %, corrected according to the NBS-22 standard with the known d13C value of -29.7 %). During each batch of analyses, a vacant Sn capsule was measured as the blank to examine the background, and a carbon black sample with the foregone d13C value of -36.9 ± 0.1 % was adopted to test the reproducibility and accuracy. In our
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study, low background, good reproducibility and high accuracy were acquired. The standard deviation of the measurements and the difference between the measured data and foregone value were accordingly less than 0.5 %.
Results and Discussion Isotopic Ratios for Fruiting Body, PE-Soluble Extract, Ergosteryl Esters and Triglycerides Ergosteryl esters and triglycerides in the PE-soluble extract were separated and purified by a preparative column filled with activated silica gel. The reversed-phase HPLC, developed previously for the simultaneous determination of free and esterified ergosterols in fungi [6], was used to monitor the PE-soluble extract from the fruiting body of G. lucidum (Fig. 1a) and ergosteryl esters purified by the CC method (Fig. 1b). After the CC separation and purification, 45.30 mg of ergosteryl esters and 921.70 mg of triglycerides were obtained from 100.03 g of the dried fruit bodies of G. lucidum. The d13C values of fruiting body, PE-soluble extract, ergosteryl esters and triglycerides from G. lucidum were determined by EA–IRMS and are shown in Table 1. Their d13C values ranged from -23.6 to -26.4 %, showing a progressive depletion in 13C from the fruiting body to triglycerides, to PE-soluble extract, and to ergosteryl esters. The utmost difference in the d13C values between the fruiting body and its PE-soluble extract was from 2.5 to 2.7 %, while the difference among the ergosteryl esters, PE-soluble extract and triglycerides was in the range of 0.5–0.7 %. There is no obvious difference in the d13C values between coarse particles and fine powders of the fruiting body (Table 1), demonstrating the homogenous distribution of stable carbon isotopes in G. lucidum. Previous studies suggested that the d13C values of fungi closely followed those of their substrates, which possibly possessed dissimilar carbon isotope ratios, and a carbon isotope fractionation might occur between fungi and their substrates [11, 12]. In this case, the comparatively homogenous d13C values and the obviously more depleted 13 C values in G. lucidum and PE extract in comparison with those of C4 plants demonstrate that the artificially Ganoderma-cultivated substrate may be attributed to C3 plants [13–15]. Fatty Acid Composition in Triglycerides and Ergosteryl Esters Fatty acid compositions in triglycerides and ergosteryl esters from G. lucidum were analyzed by gas chromatography–mass spectrometer (GC–MS) and are shown in
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Fig. 1 HPLC chromatograms of PE-soluble extract from G. lucidum (a) and ergosteryl esters purified by column chromatograph (b). Peaks: (1) ergosterol; (2) ergosteryl esters; and (3) ergosteryl esters
Table 1 d13C values (%) of the fruiting bodies of G. lucidum and its PE-soluble extract, ergosteryl esters and triglycerides
d13C (%)
Coarse fruiting body (*60 mesh)
Fine fruiting body (\120 mesh)
PE-soluble extract
Ergosteryl esters
Triglycerides
-23.6
-23.8
-26.3
-26.5
-25.8
13
13
The d C values are given in per mill. Coarse, about 60 mesh. Fine, less than 120 mesh. The d C values are the means of three determinations, and have their typical standard deviations were less than 0.3 %
Table 2. The results show that the profiles of fatty acids varied in triglycerides and ergosteryl esters (Fig. 2). Among the individual fatty acids, as can be seen from Table 2, CD9c 18:1 was the predominant fatty acid and constituted 61.26 % of the total fatty acids while C16:0 (15.81 %), CD9c,12c (13.69 %) and C18:0 (3.67 %) were the secondary 18:2 fatty acids in triglycerides from G. lucidum. The result was in accordance with the previous results, showing that C18:1, C16:0, C18:2 and C18:0 are the main constituents in the lipids of Ganoderma spores [10]. Kalacˇ [16] identified tens of fatty acids in mushroom lipids and showed that C18:2 and C18:1 markedly prevailed, forming a proportion of twothirds and more of the weight of all identified fatty acids, while saturated C16:0 was third in order. Rupcˇic´ and Juresˇic´ [7] showed that the neutral lipid fraction contained mainly C16:0, C16:1, C18:0 and C18:1, and C16 fatty acids constituted
about 60 % of total fatty acids. Ferreira et al. [17] suggested that triglycerides displayed a limited capacity for the storage of saturated fatty acids, even under conditions of saturated fatty acid accumulation. It was reported that triglycerides synthesized in the absence of oxygen were specifically enriched in C16:0, which is the main saturated fatty acid [17]. The excess saturated fatty acids are not principally stored in triglycerides and steryl esters but in special phospholipid species, especially phosphatidylinositol [7, 17]. However, the present results showed that the proportion of saturated fatty acids in ergosteryl esters (84.22 %) was notably higher than that in triglycerides (22.29 %). In contrast to fatty acids in triglycerides, C16:0 was the predominant fatty acid and constituted 71.88 % of the total D9c,12c fatty acids while CD9c (5.91 %) were 18:1 (9.12 %) and C18:2
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Table 2 Relative contents of fatty acids in ergosteryl esters and triglycerides from the fruiting bodies of G. lucidum Fatty acids
Triglycerides (%)
Ergosteryl esters (%)
C12:0
0.26
ND
C14:0
0.07
0.13
C15:0
0.70
2.09
C16:0
15.81
71.88
CD9c 16:1
0.16
0.14
CD7c 16:1
1.29
0.25
C17:0 CD11c 17:1
0.25 0.17
1.69 ND
C18:0
3.67
2.41
CD9c 18:1
61.26
9.12
CD8c 18:1
0.58
0.35
CD9c,12c 18:2
13.69
5.91
C20:0
0.42
0.12
CD11c 20:1
0.56
0.11
C22:0
1.13
1.07
C23:0
ND
0.56
C24:0
ND
4.28
Rsat
22.29
84.22
Rmonounsat
64.02
9.95
Rpolyunsat
13.69
5.91
Runsat
77.71
15.86
Rsat/Runsat
0.29
5.31
sat, monounsat, polyunsat and unsat represent saturated, monosaturated, polyunsaturated and unsaturated fatty acids, respectively. ND no detection
the secondary fatty acids in ergosteryl esters from G. lucidum. It has been reported that the principal fatty acids of steryl esters in yeast were C16 and C18 unsaturated fatty acids [8]. Ferreira et al. [17] indicated that saturated fatty acids cannot be efficiently stored within steryl esters and suggested that the absence of sterol moieties as saturated fatty acid counterparts for steryl ester synthesis can be accounted for by ergosterol biosynthesis shutdown. An accumulation of unesterified sterol in cell membranes would probably be deleterious [18]. Therefore, a surplus of free sterols in the endoplasmic reticulum is alleviated via esterification with fatty acids by two acyltransferases to maintain intracellular free sterol at nontoxic concentrations [1]. When the biosynthesis of ergosterol increases, the esterification of ergosterols additionally needs an increased quantity of fatty acids for the storage of ergosterols in cells and the biosynthesis of fatty acids increases [19]. Although being unnecessary for fungal viability, steryl esters storage and mobilization contribute markedly to sterol homeostasis and distribution [3]. The regulation of synthesis and eduction from the endoplasmic reticulum are adequate to preclude the accretion of free sterols in the endoplasmic reticulum [1].
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Fig. 2 GC chromatograms of fatty acids in triglycerides (a) and ergosteryl esters (b) from G. lucidum. Main peaks: (1) C16:0; (2) C18:0; (3) C18:1; and (4) C18:2
In the absence of oxygen, fungal cells transfer ergosterol from the plasma membrane to the endoplasmic reticulum, in which the free ergosterol is esterified and then stored in lipid particles [1]. It is possible that, after fungal death, free ergosterols in the cell membrane of the dead fungus were esterified with preferentially saturated fatty acids (mainly C16:0). A previous study had shown that free ergosterol in the dead fungi underwent esterification to be stored in lipid particles for a longer period while free ergosterol markedly decreased [6], indicating that the free and esterified ergosterols differ in their decomposition rates in the dead fungi and the esterified ergosterols are more stable than the free form [4, 6, 20]. Wallander et al. [21] found that, in freshly generated mycelia, all the ergosterol existed nearly in free form, and in the old soil organic matter from the mineral layer, free ergosterol made up only 20 % of total ergosterols, indicating that a large portion of free ergosterol was esterified in older soil organic matter, and this portion was likely to be related with dead fungi. Recently, Clemmensen et al. [22] showed that the proportion of esterified ergosterol increased during mycelial senescence. Isotopic Ratios for Individual Fatty Acids in Triglycerides and Ergosteryl Esters The d13C values of individual main fatty acids in triglycerides and ergosteryl esters from G. lucidum were
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Table 3 d13C values (%) of major fatty acids in triglycerides and ergosteryl esters from the fruiting bodies of G. lucidum Fatty acids
d13C (%) Triglycerides
Ergosteryl esters
C16:0
-25.8
-26.8
C18:0
-24.8
-26.0
C18:1 C18:2
-25.6 -25.3
-26.5 -26.6
The d13C values are the means of three determinations, and have typical standard deviations of less than 0.3 % (SDC16:0 0:15 &; SDC18:0 0:27 &; SDC18:1 0:19 &; and SDC18:2 0:23 &)
determined by GC–IRMS and corrected according to Eq. 2 and are shown in Table 3. As can be seen from Table 3, the main fatty acids C16:0, C18:0, C18:1 and C18:2 in triglycerides and ergosteryl esters have the d13C values within the ranges of -25.8 to -24.8 % and -26.8 to -26.0 %, respectively, disclosing the relative uniformity within themselves but evident difference between them in an analytical error of ±0.5 %. The main fatty acids in ergosteryl esters may fully be derived directly from triglycerides, due to triglycerides as one depot fat with such a large quantity relative to ergosteryl esters in G. lucidum. The d13C values of main fatty acids in ergosteryl esters exhibit a pronounced depletion in 13C compared with triglycerides within the range of -1.3 to -0.9 %, preferably supporting the above inference. Free ergosterol in the dead fungi is esterified with fatty acids derived from triglycerides, mainly saturated fatty acids such as C16:0 and then stored in lipid particles during the death of fungal hyphae. During the process, triglycerides may be partially hydrolyzed to fatty acids and diacylglycerol, which serves primarily as an acceptor for the esterification of fatty acids and does not show membrane-damaging feature [19, 23]. The C16:0 fatty acid in triglycerides from G. lucidum may be the basic material for desaturation and elongation to longer-chain length fatty acids, and may play a principal role in the synthesis of additional fatty acids in G. lucidum [10, 24]. Theoretically, both elongation and desaturation of the fatty acids in G. lucidum may result in the 13C depletion compared with their precursors, even if no carbon atoms are involved in desaturation, because the addition of one neutron can reduce the rate of a chemical reaction considerably [24]. A previous study [10] showed that the average d13C values of major fatty acids in triglycerides are characterized by d13C16:0 \ d13C18:0 [ d13C18:1 & d13C18:2 (Table 3), i.e., during the carbon chain elongation, C18:0 has an obvious enrichment in 13C in comparison with C16:0 within a range of ?1.0 %, the desaturation of C18:0 to C18:1 conduces to a 13C depletion of -0.8 %; but the subsequent desaturation from C18:1 to C18:2 exhibits no obvious 13C fractionation of
?0.3 %. Obviously, the variation of d13C values in the desaturation from C18:0 to C18:1 and further to C18:2 are fully or basically consistent with the above prognosis, while an evident 13C enrichment abnormally occurs in the elongation from C16:0 to C18:0. This abnormality may be attributed to the massive biosynthesis of fatty acid C18:1 in G. lucidum, i.e., although a 13C depletion may occur in the elongation from C16:0 to C18:0, the plentifully biosynthesized C18:1 may counterbalance this 13C depletion, and even produce a remarkable 13C enrichment in C18:0 just as in this case (-24.8 %) (Table 3). The results have illustrated the importance of clarifying the metabolism and biosynthesis (the elongation and desaturation of chain) of fatty acids, which cause the 13C/12C discrimination detected in triglycerides [25, 26]. In conclusion, fatty acid compositions in the triglycerides and ergosteryl esters from the fruiting body of G. lucidum were analyzed by GC–MS and the results showed that the profiles of fatty acids varied in triglycerides and ergosteryl esters. The percent of saturated fatty acids in ergosteryl esters was remarkably higher than that in triglycerides. CD9c 18:1 was the predominant fatty acid and constituted 61.26 % of the total fatty acids while C16:0, CD9c,12c and C18:0 were the secondary fatty acids in tri18:2 glycerides. In contrast to fatty acids in triglycerides, C16:0 was the predominant fatty acid and constituted 71.88 % of D9c,12c the total fatty acids while CD9c were the 18:1 and C18:2 secondary fatty acids in ergosteryl esters. It is probable that, after the fungal death, free ergosterol in the dead fungi is esterified with preferentially saturated fatty acids such as C16:0 and then stored in lipid particles for a longer period while free ergosterol markedly decreases. The d13C values of main fatty acids in ergosteryl esters exhibit a pronounced depletion in 13C in comparison with triglycerides within a range of -1.3 to -0.9 %, preferably supporting the above inference. Acknowledgments This work was co-supported by the Ph.D. Programs Foundation of the Ministry of Education of China (20090171110015) and the National Key Basic Research Program of China (973 program) (No.: 2012CB956004). We thank Mr. Shi-Ping Xu at the Guangzhou Institute of Geochemistry, Chinese Academy of Sciences for his experimental assistance.
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