8
Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues Noem ı Ruiz-L opez, Enrique Mart ınez-Force, and Rafael Garc es * Instituto de la Grasa, Consejo Superior de Investigaciones Cient ıficas, E-41012 Seville, Spain Received 27 December 2002 Abstract A method for plant tissue digestion and triacylglycerol (TAG) extraction followed by transmethylation of TAGs to produce the fatty acid methyl esters (FAMEs) from small storage tissue samples is presented. The method allows the analysis of both TAGs and FAMEs from the same sample. Several reagent mixtures and different experimental conditions were tested on sliced sunflower seeds. The best results were obtained using a mixture that was 33.3% a solution of NaCl (0.17 M) in methanol and 66.6% heptane by volume. The TAGs in the heptane solution were transmethylated with a mixture containing methanol:toluene:dimethoxypropane:H 4 SO 2 (39:20:5:2, by vol). The method was also tested on other oil seed storage tissue (soybean) and fruit tissues from olive and acorn. In all cases, sunflower, soybean, olive, and acorn, the TAGs and FAMEs composition data obtained by this method were quite similar to data from a standard analysis method. In samples with high protein content, such as soybean and sunflower seeds, the TAG extraction was incomplete. The water content of fruit samples did not interfere with TAG extraction obtained by this method. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Triacylglycerols; Fatty acids; Capillary gas chromatography; Extraction The chemical, physical, and nutritional properties of oils are affected by triacylglycerol (TAG) 1 composition and their stereospecificity (position of the fatty acids (FA) on the glycerol backbone). Nevertheless the levels of mi- nor constituents are also important, for example in olive oil [1,2]. TAG composition gives more information about a particular oil (like a fingerprint). Current methods of oil extraction and analysis are time-consuming and imprac- tical for processing a high number of samples. Thus, it would be very useful to develop a method that allowed a direct extraction of lipids from a small piece of seed, in order to analyze TAG composition and to transmethylate FAs later. This method would allow the selection of in- dividual oleaginous seeds from a wide collection on the basis of their TAG composition or to characterize the TAG composition of a high number of samples. Several methods have been used to extract oil from seed samples. The AOAC-accepted reflux–extraction procedures, such as Goldfisch and Soxhlet, are not suitable for a large number of small samples. Other methods of total lipid extraction could be used for a relatively high number of samples, but these methods involve several steps. Folch et al. [4] devised a method for total lipid extraction using a solution of chloro- form:methanol (2:1, v/v) that was originally used for the extraction of brain tissue lipids. Some modifications were developed by Wren et al. [5], Dawson et al. [6], and Privett et al. [7], making the method more suitable for specific tissues. Hara and Radin [8] introduced a new method with no toxic solvents using hexane:isopropanol (3:2, v/v). These multistep methods could be used for small samples, but not for a large number of them. Reske et al. [9] used a method in which approximately 100 sunflower seeds were ground with hexane in a 1:1 (w/v) ratio and stood overnight. Chaven et al. [10] proposed a relatively simple microanalytical technique for isolating the neutral lipid fraction of soybean lipids and analyzing their FA composition. The microanalyt- ical procedure was suitable for small samples but like the Analytical Biochemistry 317 (2003) 247–254 www.elsevier.com/locate/yabio ANALYTICAL BIOCHEMISTRY * Corresponding author. Fax: +34-954-616790. E-mail address: [email protected] (R. Garc es). 1 Abbreviations used: BHT, butylated hydroxytoluene; DMP, 2,2- dimethoxypropane; FA, fatty acid; FAME, fatty acid methyl ester; L, linoleic acid (18:2); MeOH, methanol; HHH, triheptadecanoylglycerol; O, oleic acid (18:1); P, palmitic acid (16:0); S, stearic acid (18:0); TAG, triacylglycerol. 0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00139-8

Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues

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Page 1: Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues

Sequential one-step extraction and analysis of triacylglycerolsand fatty acids in plant tissues

Noem�ıı Ruiz-L�oopez, Enrique Mart�ıınez-Force, and Rafael Garc�ees*

Instituto de la Grasa, Consejo Superior de Investigaciones Cient�ııficas, E-41012 Seville, Spain

Received 27 December 2002

Abstract

A method for plant tissue digestion and triacylglycerol (TAG) extraction followed by transmethylation of TAGs to produce the

fatty acid methyl esters (FAMEs) from small storage tissue samples is presented. The method allows the analysis of both TAGs and

FAMEs from the same sample. Several reagent mixtures and different experimental conditions were tested on sliced sunflower seeds.

The best results were obtained using a mixture that was 33.3% a solution of NaCl (0.17M) in methanol and 66.6% heptane by volume.

The TAGs in the heptane solution were transmethylated with a mixture containing methanol:toluene:dimethoxypropane:H4SO2

(39:20:5:2, by vol). The method was also tested on other oil seed storage tissue (soybean) and fruit tissues from olive and acorn. In all

cases, sunflower, soybean, olive, and acorn, the TAGs and FAMEs composition data obtained by this method were quite similar to

data from a standard analysis method. In samples with high protein content, such as soybean and sunflower seeds, the TAG

extraction was incomplete. The water content of fruit samples did not interfere with TAG extraction obtained by this method.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: Triacylglycerols; Fatty acids; Capillary gas chromatography; Extraction

The chemical, physical, and nutritional properties of

oils are affected by triacylglycerol (TAG)1 composition

and their stereospecificity (position of the fatty acids (FA)

on the glycerol backbone). Nevertheless the levels of mi-

nor constituents are also important, for example in olive

oil [1,2]. TAG composition gives more information about

a particular oil (like a fingerprint). Current methods of oilextraction and analysis are time-consuming and imprac-

tical for processing a high number of samples. Thus, it

would be very useful to develop a method that allowed a

direct extraction of lipids from a small piece of seed, in

order to analyze TAG composition and to transmethylate

FAs later. This method would allow the selection of in-

dividual oleaginous seeds from a wide collection on the

basis of their TAG composition or to characterize theTAG composition of a high number of samples.

Several methods have been used to extract oil from

seed samples. The AOAC-accepted reflux–extraction

procedures, such as Goldfisch and Soxhlet, are not

suitable for a large number of small samples. Other

methods of total lipid extraction could be used for a

relatively high number of samples, but these methods

involve several steps. Folch et al. [4] devised a methodfor total lipid extraction using a solution of chloro-

form:methanol (2:1, v/v) that was originally used for the

extraction of brain tissue lipids. Some modifications

were developed by Wren et al. [5], Dawson et al. [6], and

Privett et al. [7], making the method more suitable for

specific tissues. Hara and Radin [8] introduced a new

method with no toxic solvents using hexane:isopropanol

(3:2, v/v). These multistep methods could be used forsmall samples, but not for a large number of them.

Reske et al. [9] used a method in which approximately

100 sunflower seeds were ground with hexane in a 1:1

(w/v) ratio and stood overnight. Chaven et al. [10]

proposed a relatively simple microanalytical technique

for isolating the neutral lipid fraction of soybean lipids

and analyzing their FA composition. The microanalyt-

ical procedure was suitable for small samples but like the

Analytical Biochemistry 317 (2003) 247–254

www.elsevier.com/locate/yabio

ANALYTICAL

BIOCHEMISTRY

* Corresponding author. Fax: +34-954-616790.

E-mail address: [email protected] (R. Garc�ees).1 Abbreviations used: BHT, butylated hydroxytoluene; DMP, 2,2-

dimethoxypropane; FA, fatty acid; FAME, fatty acid methyl ester; L,

linoleic acid (18:2); MeOH, methanol; HHH, triheptadecanoylglycerol;

O, oleic acid (18:1); P, palmitic acid (16:0); S, stearic acid (18:0); TAG,

triacylglycerol.

0003-2697/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0003-2697(03)00139-8

Page 2: Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues

previous techniques still required separate steps forextraction, filtration, etc.

There are several methods for determination of

composition of isolated TAGs: thin-layer chromatogra-

phy (TLC), high-performance liquid chromatography

(HPLC), gas–liquid chromatography (GLC), and super-

critical fluid chromatography. The recommended IU-

PAC method uses isocratic nonaqueous reversed-phase

high-performance liquid chromatography, with refrac-tive index detection, rendering separations based on the

equivalent carbon number. On the other hand,GLC gives

a very high resolution and is able to separate TAGs with

saturated FAs that have poor solubility and long reten-

tion times in HPLC with refractive index detection. GLC

allows the analysis of very complex mixtures of TAGs

with many different molecular weights [11].

The TAG composition can be calculated utilizing fattyacid methyl ester (FAME) determinations and computer

programs, by applying the 1,3-random 2-random distri-

bution theory [12]. For this purpose a lipase digestion is

made and resulting lipids are separated by TLC. The

monoacylglycerol band containing the FAs in position

sn-2 in TAG molecules and the total FA composition in

TAG molecules are used with a computer program, LI-

PASE, to calculate the original TAG composition (http://niobio.grasa.csic.es/emforce/GGBLS/). Nevertheless this

method is suitable only to get a reference of the TAGs that

should be found in any oil sample, because of the asym-

metrical distribution of some FAs in the sn-1 and sn-3

positions of the TAGs [9,11].

With the aim of analyzing the FA composition of

several thousands of individual half-seed samples in

mutagenized sunflower seeds, a method for direct oilextraction and FA transmethylation was developed [13].

This method used a complex reagent mixture that di-

gested seed tissues, extracted the lipid fraction, trans-

methylated the FAs, and separated these esters from the

rest of the components in just one step.

In this work, we describe a new analytical method to

extract oil from seed samples in one step, analyze TAG

composition by GLC, transmethylate the FAs in thesample later, and analyze the FAME composition by

GLC. This allows the analysis of a large number of very

small samples like half-seeds. Because the embryo part

of the seed is not used for the analysis, it could be ger-

minated and the descendants obtained in case the de-

sired phenotype is found in a screening of mutagenized

or segregant seeds.

Materials and methods

Plant materials and growth conditions

Sunflower (Helianthus annuus L.) seeds from the high-

oleic line CAS-9 [11] and the medium stearic line CAS-4

[3] were used in this work. Seeds were collected fromplants grown in a growth chamber at 25/15 �C day/night,

16-h photoperiod, and 300lE �m�2 � s�1 light intensity.

Seeds were immersed in water for 2 h to facilitate peeling

and the achenes (seeds without seed coat) were dried and

cut into pieces. Seed pieces were placed in screw-cap test

tubes with Teflon-lined caps. For the nondestructive

half-seed method, one-third of the cotyledon (opposite

the embryo side) was analyzed to get TAG and FAMEcomposition. The rest of the seed, containing the em-

bryo, could be grown and the progeny collected. Sample

sizes are indicated in each experiment. Olive (Olea

europea) fruits, acorn (Quercus ilex) fruits, and soybean

(Glycine max) seeds were also used to test the method.

Solutions and reagents

1,2,3-Triheptadecanoylglycerol (HHH) and butylated

hydroxytoluene (BHT) were from Sigma–Aldrich Che-

mie, Gmbh (Steinheim, Germany). Hexane, heptane,

isopropanol, methanol, tethahydrofuran, and washed

sea sand were obtained from Panreac Qu�ıımica (Barce-

lona, Spain). 2,2-Dimethoxypropane (DMP) was ob-

tained from Merck (Darmstadt, Germany). Specific

solution mixtures are indicated in each experiment. So-lutions of 0.17M NaCl in methanol and 18.6mM HHH

in heptane (used as internal standard) were prepared

and added to all experiments. BHT at 0.01% (w/v) was

also added to all experiments as an antioxidant.

Oil extraction

Unless indicated, samples were incubated withoutshaking for 1 h in a water bath at 80 �C. Incubation

mixtures for TAG extraction are shown in Table 1.

After cooling, the upper phase containing the lipids was

transferred to a new test tube. For some experiments,

the lipids that remained in the seed pieces after the in-

cubation at 80 �C were extracted using the method of

Hara and Radin [8]. First, seed pieces were washed two

times in heptane to avoid any external lipid contami-nation. As control of total lipid content, seed pieces were

extracted with 50 ll of HHH in heptane as internal

control, see above. Three different replicates were made

of each experiment. TLC was carried out in 0.25-mm-

thick silica gel G-60 plates developed with hexane:di-

ethyl ether (90:10; v/v). To detect oil spots, plates were

sprayed with 50% H2SO4 in water and heated at 200 �Cuntil the spots were visible.

For time-course extraction experiments, different

amounts of seed pieces (200, 50, and 20mg) were incu-

bated with the selected mixture solution in a final vol-

ume of 7.2ml. After 15, 30, 60, 120, and 240min an

aliquot was sampled and analyzed for TAG extraction

with respect to the added internal standard. The seed

pieces remaining in the test tubes after the partial

248 N. Ruiz-L�oopez et al. / Analytical Biochemistry 317 (2003) 247–254

Page 3: Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues

extraction were cleaned two times with heptane and

extracted using the method of Hara and Radin [8], see

above. These time-course experiments were repeated

four times.

TAG analysis by GLC

TAG were separated and quantified by GLC [11] with

an Agilent 6890 gas chromatograph (Palo Alto, CA,

USA), hydrogen was used as carrier gas, injector and

detector temperature was 380 �C, oven temperature was

345 �C, and a gradient of pressure from 70 to 120 kPa

was used, depending on each particular column. The

gas-chromatography capillary column was a DB-17HT

(15m length, 0.25mm i.d., and 0.15 lm film thickness)with a midpolarity liquid phase of (50% phenyl)meth-

ylpolysiloxane from J&W Scientific (Folsom, CA,

USA), the linear gas rate was 50 cm/s, and the split ratio

was 1:80. The detector used was a flame ionization de-

tector. The different TAG molecules were identified with

respect to known samples [11] and the detector response

was corrected [14]. Total lipid extracted in each experi-

ment was calculated with respect to the area of the in-ternal control HHH. For TAG analysis by GLC it is not

necessary to purify the TAG from the other neutral

lipids extracted by this method in the sample because of

their very different retention times.

FAME extraction and analysis

After the analysis of TAG, the remaining heptaneTAG solution was filled to 0.9ml of total solution with

heptane, and another 0.9ml of the transmethylation

mixture containing methanol:toluene:DMP:H2SO4 (39:

20:5:2) was added [13]. The FAMEs were quantified us-

ing a Hewlett–Packard 5890A gas chromatograph (Palo

Alto, CA, USA) with a Supelco SP-2380 (Bellefonte, PA,

USA) capillary column of fused silica (30m length;

0.25mm i.d., and 0.20 lm film thickness). Hydrogen wasused as carrier gas and the linear gas rate was 28 cm/s.

Detector and injector temperatures were 200 �C, oven

temperature was 170 �C, and the split ratio was 1:50.

FAMEs were identified by comparison with known

standards. Fatty acid composition was calculated from

TAG composition assuming that each fatty acid in a

TAG molecule represents one-third of the percentage of

that molecule in the total TAGs.

Oil extraction from other plant tissues

To obtain quantitative TAG extraction data from

olive (O. europea) and acorn (Q. ilex) fruits, 50mg of

fruit pieces was incubated at 80 �C with 7.2ml of the

new method mixture and 16 ll of HHH solution as

internal standard during 2 h. After cooling, the upper

phase containing the lipids was transferred to a newtest tube. The remaining lipids in the fruit pieces were

extracted as indicated above [8], after addition of 8 ll ofHHH solution. The lipidic phases from the extracted

samples were evaporated to dryness under N2. The

residue was dissolved in a small volume of heptane.

To compare qualitative TAG extraction data from

these fruits, 50mg of the samples was treated as indi-

cated above and 50mg of the samples was treated bythe method of Hara and Radin [8] without previous

extraction.

Soybean (G. max) seeds were immersed in water for

1 h. Fifty milligrams from one individual seed was cut

and TAGs were extracted with the new extraction mix-

ture as indicated above for olive and acorn tissues. The

seed pieces remaining in the test tubes after the partial

extraction were cleaned two times with heptane andextracted [8], see above.

Results and discussion

The goal of this work was to develop a procedure for

fast and easy TAG and FAME preparation and analysis

by GLC. In the first step TAGs were extracted andanalyzed by GLC. Then, TAGs were transmethylated.

Isolated FAMEs could also be analyzed by GLC.

Table 1

Solvent mixtures by volume assayed and total TAGs extracted expressed as percentage of the total TAG content (extracted by the Hara and Radin

method)

Mixture Heptane MeOH/NaCla Toluene DMP THF HHHb % Extracted

A 61.1 33.3 — — — 5.6 79:7� 1:1

B 56.1 33.3 5.0 — — 5.6 74:2� 3:2

C 51.1 33.3 10.0 — — 5.6 70:8� 3:5

D 55.1 33.3 5.0 1.0 — 5.6 74:0� 1:5

E 54.1 33.3 5.0 2.0 — 5.6 72:6� 2:5

F 50.1 33.3 10.0 1.0 — 5.6 76:3� 4:6

G 49.1 33.3 10.0 2.0 — 5.6 70:6� 1:2

H 51.1 33.3 — — 10.0 5.6 76:3� 8:7

Extraction time was 2 h at 80 �C. Percentage of extraction is expressed as mean� SD of three replicates.aA solution of 10 g/liter of NaCl in methanol.b Triheptadecanoylglycerol (18.6 mM) in heptane.

N. Ruiz-L�oopez et al. / Analytical Biochemistry 317 (2003) 247–254 249

Page 4: Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues

This method allows the determination of TAG andFA composition of a large number of small samples

(3–5mg) in a short time.

First step—TAG extraction and analysis

In a previous work Garc�ees et al. [13] developed a

method for one-step oil extraction, FAME production,

and analysis. Following the same philosophy, our goalin this work was to perform a quick extraction of the oil

but without the transmethylation step. For this, several

solutions were designed to digest the tissue and extract

the lipids. In order to have complete extraction of lipids,

it is necessary to use organic extractants of high polarity,

usually containing some alcohol. We used solvents

containing methanol, a primary alcohol with the most

active hydroxyl group. Methanol and water stimulateddisruption of hydrogen bonds between lipid carbonyl,

hydroxyl, and amino groups and compounds of the

nonextractable residue [15]. As a nonpolar solvent we

used heptane, which has a higher boiling point than

hexane (the usual solvent). Some other chemical com-

pounds were also added to the mixture in order to test

whether the total lipid extraction was improved. Tolu-

ene or tetrahydrofuran was used, instead of benzene,because of its lower toxicity, to form a unique phase at

80 �C for lipid extraction. The addition of DMP was

also found very useful to help in the extraction of lipids

[13]. An important modification was the change of

H2SO4 to NaCl. This compound was added in order to

have two phases and a good separation of TAGs from

polar compounds at room temperature and to avoid the

production of the FAMEs because of the presence ofmethanol with acid pH. An antioxidant was added to all

mixtures in order to avoid partial oxidation of lipids.

This was especially relevant in the case of samples with a

high proportion of unsaturated FAs. We selected BHT

as antioxidant because it does not affect the polarity of

the mixture.

Table 1 shows the different mixtures we designed and

the ratio of total TAG extraction obtained after 2 h at80 �C with each mixture relative to the complete ex-

traction (100%) obtained using the method of Hara and

Radin [8]. All these mixtures have the property of

forming a single phase at 80 �C, improving extraction of

lipids, and two phases at room temperature with neutral

lipids contained in the upper phase (Fig. 1, lanes A and

B). The seeds used in this particular experiment were

from a high-oleic sunflower line because of its lownumber of TAG peaks in the chromatogram and the

peak of the internal control (HHH) being easily distin-

guishable from the rest. All the mixtures tested gave an

extraction of TAG ranging between 70 and 80%, the

best results being obtained with mixture A, which is also

the simplest. Results obtained were not better after

adding toluene, tetrahydrofuran, or DMP. Additionally,

mixtures containing tetrahydrofuran showed poor sep-

aration of phases at room temperature.

In order to verify that extraction with mixture A does

not produce any transmethylation we carried out an

experiment extracting oil from sunflower seeds. The

lipids were separated by TLC. Fig. 1 shows that the oil

extracted by our method does not produce any FAMEs(line B) but diacylglycerols are extracted (see light-gray

band); we could suggest that only neutral lipids were

extracted. Lane A shows sunflower oil lipids separated

by TLC and lane C is a partial transmethylation of

sunflower oil (see dark-gray band at the top corre-

sponding to FAMEs).

To optimize the extraction, a time-course experiment

with different sample sizes was carried out using mixtureA from 15 to 240min. Samples for these assays were

from medium-stearic sunflower line CAS-4. Total mil-

ligrams extracted from each sample is shown in Fig. 2A.

The amount of total TAG extracted increased up to

120min. In the three cases the behavior was similar,

indicating that 200mg sample did not saturate the

mixture. The amount of lipid extracted in this case was

higher than in the others with 20 and 50mg of seedsamples and was similar from 120 to 240min. Fig. 2B

shows that the extraction of lipids was very efficient,

with more than 50% of TAG extracted after 30min.

After that, the extraction slowed with no important

extraction increase up to 240min. We selected the time

of 120min, which gave near-maximum extraction under

these conditions independent of sample size.

Table 2 shows the TAG composition of the extractedoil using different sample sizes and extraction times.

Interestingly, the TAG composition is very similar in all

cases, with only a few cases exhibiting small differences

between the TAG content of two different points. The

standard deviation shows that no significant difference

could be found between the different extraction times.

Those differences might not be related to the sample size

Fig. 1. TLC plate of sunflower oil (lane A), oil extracted by mixture A

(heptane/MeOH/NaCl) for 2 h at 80 �C (lane B), and partial transme-

thylation of sunflower oil (lane C).

250 N. Ruiz-L�oopez et al. / Analytical Biochemistry 317 (2003) 247–254

Page 5: Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues

or extraction time, but reflect the intrinsic differencesbetween the TAG compositions of different seeds. It is

important to note that the seed samples of 200mg, when

only 30% of TAGs were extracted, have the same TAGmolecular species content as the smallest, 20-mg seed

samples. These observations indicate that this method

gives consistent results with any extraction time and

sample size. Thus, we suggest using a sample size of

around 20 to 50mg and an extraction time of 120min,

leading to a precise TAG composition and efficient ex-

traction.

Finally, we verified the accuracy of the new extractionmethod by analyzing the TAG composition of the two

cotyledons obtained by transversally slicing an individ-

ual seed, having two identical samples and avoiding in

this case the intrinsic seed-to-seed TAG composition

differences. Each cotyledon was analyzed by extracting

the oil with mixture A (Table 1) and/or the control

method [8] and the TAG composition was compared.

Table 3 confirms that there is seed-to-seed variation,comparing the results obtained by either method be-

tween any of the seeds tested. For example, in the two

comparisons of the control method (seeds A and B) the

POP contents for each cotyledon of seed A were 0.48

and 0.51 and in seed B the values were 0.40 and 0.39. In

this case there is around 10% difference between seeds A

and B, but in the case of seeds A and C the POO con-

tents were 5.10 and 5.03 for seed A and 2.74 and 2.65 forseed C much lower than for seed A. Similar results were

obtained with the other TAG and seed method com-

parisons. In any case, it is relevant that the TAG com-

positions determined in both halves of any seed are very

similar, indicating that the new method precisely reflects

the real TAG composition of the samples.

Table 2

Molecular species of CAS-4 line TAG (mol%) found in the upper phase (heptane) using mixture A of Table 1 at 15 (A), 60 (B), and 240min (C) of

incubation and the remaining TAG found in these seeds extracted with the control method (D)

TAG type Molecular TAG speciesa (mol%)

HR 20mg 50mg 200mg

Mean�SD A B C D SD A B C D SD A B C D SD

POP 0:3� 0:0 0.3 0.4 0.3 0.3 0.1 0.4 0.3 0.3 0.3 0.1 0.3 0.3 0.3 0.3 0.0

PLP 0:5� 0:0 0.5 0.6 0.5 0.6 0.1 0.6 0.5 0.5 0.6 0.1 0.5 0.5 0.5 0.5 0.0

POS 1:5� 0:2 1.8 1.5 1.4 1.4 0.3 1.4 1.4 1.3 1.5 0.1 1.6 1.4 1.4 1.5 0.1

POO 2:8� 0:2 3.2 2.9 2.6 2.7 0.3 2.8 2.8 2.6 2.6 0.2 3.0 2.6 2.7 2.7 0.2

PLS 2:5� 0:2 2.9 2.7 2.7 2.9 0.3 2.6 2.6 2.6 2.7 0.1 2.6 2.5 2.5 2.6 0.1

POL 5:3� 0:1 6.9 6.4 5.8 5.8 0.5 5.8 5.8 5.9 5.7 0.2 5.5 5.3 5.5 5.4 0.1

PLL 4:2� 0:3 4.6 4.8 5.1 4.7 0.3 4.4 4.8 4.9 5.0 0.3 4.1 4.2 4.3 4.2 0.3

SOS 1:6� 0:1 1.5 1.5 1.3 1.4 0.3 1.4 1.3 1.2 1.3 0.1 1.6 1.4 1.5 1.6 0.2

SOO 7:2� 0:7 7.9 6.9 6.1 6.5 0.9 6.5 6.3 6.1 6.1 0.6 7.5 6.7 6.7 6.9 0.5

SLS 1:7� 0:2 2.4 2.2 2.0 2.3 0.4 1.8 1.8 2.1 1.8 0.3 1.9 2.7 2.0 1.8 0.7

OOO 7:0� 0:6 7.2 6.6 5.6 6.3 0.7 6.5 6.5 6.0 6.0 0.5 7.4 5.3 6.5 6.5 1.2

SOL 12:8� 0:4 12.0 12.0 11.7 12.4 0.6 12.1 11.9 11.8 11.8 0.4 12.9 12.8 12.4 12.8 0.6

OOL 14:1� 0:7 12.1 12.2 12.2 12.5 0.6 13.1 13.1 12.8 12.7 0.7 13.5 13.5 13.6 13.5 0.8

SLL 11:5� 0:6 10.2 11.3 12.6 13.1 1.1 11.9 11.7 12.1 12.4 0.7 11.2 12.1 11.9 11.9 0.6

OLL 17:2� 0:9 16.2 17.3 18.7 17.6 1.3 18.3 18.3 18.8 18.7 0.7 16.9 18.2 18.0 17.7 0.9

LLL 9:8� 1:0 10.2 10.9 11.4 9.4 1.2 10.5 11.0 11.0 10.9 1.1 9.4 10.6 10.3 10.1 1.3

The last column of each sample size experiment represents the standard deviation (SD) of the four TAG composition A, B, C, and D data. The

TAG molecular species extracted by the control method (HR) are also shown. The order of the fatty acids in the TAG type does not indicate the

TAG structure (POS¼PSO¼SPO).a P, palmitic acid (16:0); S, stearic acid (18:0); O, oleic acid (18:1); L, linoleic acid (18:2).

Fig. 2. Time-course experiment of TAG extraction with mixture A

(see Table 1) using seed samples of 20 (N), 50 (j), and 200mg (r).

Three replicates were made in each experiment. TAG extraction is

expressed as milligrams (A) and percentage (B) of total TAG calcu-

lated by the extraction method of Hara and Radin [8].

N. Ruiz-L�oopez et al. / Analytical Biochemistry 317 (2003) 247–254 251

Page 6: Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues

Second step—FAME obtainment and analysis

As previously shown, mixture A can be used to an-

alyze the TAG composition of a half-seed sample (3 to

10mg cotyledon weight). After the analysis of the TAG

composition of an aliquot of the extracted oil, the re-

maining TAG-containing heptane solution can be

transmethylated by the method of Garc�ees et al. [13] andthe FAMEs analyzed by GLC. The heptane upper phase

is transferred to a new test tube, and filled with heptane

up to 0.9ml total volume, and 0.9ml of a solution

containing methanol:toluene:DMP:H4SO2 (39:20:5:2) is

added. The tube is closed and incubated in a water bath

at 80 �C for 1 h. At 80 �C a single phase is formed and

the FAs in the sample are transmethylated. After cool-

ing, two phases separate and the FAMEs contained inthe upper phase are analyzed by GLC.

In Table 4 the calculated FA composition according

to the TAG composition obtained in the first step is

compared to the FA composition derived from FAME

analyses. The FA compositions obtained by both meth-

ods are very similar, indicating that both of them can be

used to calculate the FA composition of simple samples.

However, this second step could be useful to identify the

TAGs in some complex samples, like those from sun-flower mutant lines CAS-5 and CAS-12, that have high

palmitic content and some special TAG-containing pal-

mitoleic, asclepic, and palmitolinoleic acids [11].

Other plant tissues

In order to validate our method we tested the oil

extraction and analysis on other types of vegetablesamples with different water, fat, and protein contents,

Table 4

Fatty acid composition calculated from the TAG composition, taking into account that each TAG molecule has three fatty acids, and from analysis

by GLC of the FAMEs obtained after transmethylation of the TAG

Half-seed

sample

FA from TAG data (mol%) FAMEs from GLC (mol%) Ratio between TAG/GLC data

P S O L P S O L P S O L

A 7.3 10.4 26.0 56.3 7.1 10.6 25.6 56.7 1.03 0.98 1.02 0.99

B 7.1 11.2 23.3 58.4 7.8 10.8 22.4 59.0 0.91 1.04 1.04 0.99

C 7.2 11.1 24.9 56.8 7.2 10.9 23.8 58.0 1.01 1.01 1.04 0.98

D 6.2 13.5 33.1 47.2 6.7 13.8 31.9 47.6 0.92 0.98 1.04 0.99

E 6.5 15.1 40.7 37.8 6.7 14.8 39.7 38.8 0.98 1.01 1.02 0.97

F 6.9 15.0 27.4 50.7 7.2 14.5 27.4 50.9 0.96 1.04 1.00 1.00

Mean 6.9 12.7 29.2 51.2 7.1 12.6 28.5 51.8 0.97 1.01 1.03 0.99

SD 0.4 1.9 6.0 7.1 0.4 1.8 5.8 7.1 0.04 0.02 0.01 0.01

Six half-seeds from mutant line CAS-4 were used in this experiment (A to F). In the last two rows the means and SD of the six seed samples are

shown. Abbreviations as in Table 2.

Table 3

TAG molecular species composition (mol%) from the analysis of twin cotyledon samples obtained by cutting one seed transversally

TAG type Seed A Seed B Seed C Seed D Seed E Seed F

HR HR HR HR HR NM HR NM NM NM NM NM

POP 0.5 0.5 0.4 0.4 0.3 0.3 0.6 0.6 0.5 0.5 0.5 0.5

PLP 0.4 0.4 0.6 0.5 0.6 0.6 0.5 0.5 0.7 0.7 0.4 0.4

POS 2.3 2.5 1.9 1.9 1.9 1.8 3.0 3.0 2.0 2.0 2.4 2.2

POO 5.1 5.0 3.8 3.8 2.7 2.7 5.1 4.9 3.8 3.5 5.0 5.1

PLS 1.8 2.0 3.0 2.7 3.5 3.5 2.5 2.5 2.9 3.1 2.0 1.8

POL 5.6 5.8 6.2 6.0 5.5 5.5 6.1 6.0 6.6 6.4 5.8 5.8

PLL 2.2 2.4 4.1 3.7 4.3 4.5 2.6 2.7 4.4 4.5 2.4 2.3

SOS 2.2 2.5 1.8 1.9 2.2 2.1 3.1 3.2 1.8 1.9 2.4 2.2

SOO 12.3 12.9 9.3 9.6 8.0 7.8 12.9 13.1 8.6 8.4 12.7 12.3

SLS 1.3 1.4 2.2 2.2 3.7 3.7 2.3 2.1 1.9 2.1 1.3 1.4

OOO 13.3 12.0 8.1 8.6 4.9 5.0 10.1 9.7 7.4 6.4 12.5 13.5

SOL 12.6 13.4 13.3 13.3 15.1 14.7 14.8 14.9 13.0 13.2 13.2 12.6

OOL 19.0 17.7 14.4 15.1 11.7 11.4 15.2 15.1 14.8 13.7 17.7 18.8

SLL 6.1 6.4 11.2 10.8 14.8 15.0 7.6 7.6 10.4 11.3 6.4 5.8

OLL 12.4 12.0 14.7 15.0 14.9 14.8 10.7 10.9 15.6 15.6 12.0 12.3

LLL 2.9 3.1 5.0 4.6 5.9 6.6 2.9 3.3 5.7 6.8 3.2 3.2

Mixture A of Table 1 was used in the new method (NM) or control method (HR). Each seed (from seed A to F) was divided into the two

cotyledons cutting across the embryo; each cotyledon was analyzed by the indicated method. The experiments were carried out twice. The order of

the fatty acids in the TAG type does not indicate the TAG structure (POS¼PSO¼ SPO). Abbreviations as in Table 2.

252 N. Ruiz-L�oopez et al. / Analytical Biochemistry 317 (2003) 247–254

Page 7: Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues

such as wild olive (O. europea) fruits (high water, me-

dium fat, and low protein contents), acorn (Q. ilex)

fruits (high water and low fat and protein contents), and

soybean (G. max) seeds (very low water, low fat, and

high protein contents). Results are shown in Table 5

(olive and acorn fruits) and Table 6 (soybean seeds).

Table 5

Twin experiments comparing the TAG molecular species composition (mol%) obtained by mixture A of Table 1 (NM) and the control method (HR),

for olive and acorn fruits

TAG typea Wild olive Acorn

NM HR NM HR

Mean SD Mean SD Mean SD Mean SD

POP 4.5 0.8 4.5 0.7 4.2 0.2 4.6 0.2

PLP 2.5 0.6 2.3 0.3 4.3 0.4 5.1 0.2

PPoL/PLnP 0.4 0.3 0.3 0.1 0.3 0.4 — 0.0

POS 0.8 0.1 0.9 0.1 1.1 0.2 0.8 0.1

POO 23.3 1.4 23.7 1.4 20.0 1.3 20.7 0.5

PoOS/PLS 1.6 0.3 1.6 0.5 1.0 0.2 1.2 0.1

POL 10.6 0.8 9.9 0.7 13.6 0.7 15.1 0.2

PLL 1.7 0.3 1.5 0.1 10.1 0.7 11.2 0.5

PoOL/PLLn 0.6 0.5 0.6 0.2 1.0 0.2 0.9 0.2

SOO 2.3 0.3 2.6 0.1 2.3 0.1 1.8 0.2

OOO 31.3 2.8 32.8 1.6 18.2 1.6 17.4 0.2

SOL 4.1 0.5 4.2 0.1 2.3 0.5 1.6 0.3

OOL 12.4 2.0 11.8 2.3 9.8 0.3 8.9 0.6

SLL 1.1 0.3 0.9 0.3 1.3 0.4 0.9 0.1

OLL 2.3 0.5 2.0 0.5 7.4 0.3 7.2 0.1

OLLn 0.6 0.3 0.5 0.2 — — — —

LLL — — — — 3.2 0.6 2.8 0.2

Extracted (%) 96.8 3.2 — — 98.7 1.3 — —

In each case a sample was divided into two aliquots and each aliquot analyzed by one method. Three replicates were made. The last row shows the

means and SD of the total TAGs extracted in four experiments. The order of the fatty acids in the TAG type does not indicate the TAG structure

(POS¼PSO¼ SPO). Abbreviations as in Table 2.a Po, palmitoleic acid (16:1); Ln, linolenic acid (18:3).

Table 6

Twin experiments comparing the TAG molecular species composition (mol%) of individual soybean seeds obtained using mixture A of Table 1 (NM)

and the control method (HR)

TAG type Seed A Seed B

NM HR NM/HR NM HR NM/HR

POP 0.5 0.5 1.0 0.5 0.6 1.0

PLP 1.7 1.5 1.1 2.0 2.1 1.0

POS 0.4 0.4 1.0 0.3 0.4 0.8

POO 2.1 2.4 0.9 2.0 2.2 0.9

PLS 1.2 1.2 1.1 1.3 1.5 0.9

POL 7.8 7.9 1.0 8.6 8.8 1.0

PLL 12.9 11.8 1.1 13.6 13.3 1.0

PLLn 2.0 1.8 1.1 2.7 2.6 1.0

SOS — 0.1 — — 0.1 —

SOO 0.5 0.7 0.7 0.4 0.6 0.8

OOO 2.0 2.5 0.8 1.7 1.8 0.9

SOL 2.5 2.8 0.9 2.4 2.8 0.9

OOL 7.9 9.1 0.9 7.3 7.4 1.0

SLL 3.9 4.0 1.0 3.4 4.0 0.8

OLL 19.4 20.5 0.9 19.3 19.2 1.0

LLL 29.3 27.7 1.1 27.6 26.3 1.1

LLLn 5.8 5.2 1.1 6.7 6.4 1.1

Extracted (%) 48.6 52.8

In each case, lipids from the upper phase (heptane) were extracted by the new method described and the remaining unextracted lipids were

reextracted with the control method. The TAG composition of each lipid sample was analyzed by GLC. In the last row the total TAGs extracted by

the new method is shown. The order of the fatty acids in the TAG type does not indicate the TAG structure (POS¼PSO¼ SPO). Abbreviations as in

Tables 2 and 5.

N. Ruiz-L�oopez et al. / Analytical Biochemistry 317 (2003) 247–254 253

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The TAG extraction from olive and acorn fruits wasnear 100% when the method described in this work was

applied (see last row of Table 5), so it was not feasible to

reextract and test the TAGs remaining in the fruits using

the control method [8]. Thus, with the aim of comparing

the TAG compositions obtained by both methods in the

same samples, the fruits were cut in small pieces. Each

finely chopped sample was separated into two aliquots.

These aliquots were extracted and analyzed separatelyby the new or the control method. The TAG composi-

tions obtained by both methods were very similar (Table

5). These results show that the developed method can be

used for a quick TAG extraction and analysis in these

samples.

In experiments carried out with soybean seeds, the

new method extraction ratios were around 50% (see final

row of Table 6). Thus, to compare the TAG molecularspecies compositions obtained by both methods a sam-

ple of the seed was first extracted by the method de-

scribed in this work. The TAGs remaining in this sample

were extracted by the control method [8]. In the same

sample, TAG compositions obtained by both methods

were quite similar. Data from two individual soybean

seeds are shown (Table 6). Like for the other type of

vegetable samples, this method can be useful for thiskind of determination.

Taking into account that the extraction ratio in soy-

bean was smaller, around 50%, compared to olive and

acorn ratios, it could be suggested that the percentage of

extraction applying the new method is inversely pro-

portional to the sample protein content and that the

water content is not unfavorable for the TAG extraction.

Conclusions

In this paper we propose an easy and reliable method

for TAG and FAME sequential extraction and analysis

that is suitable for a large number of small vegetable

storage tissue samples. The best mixture contains by

volume 33.3% a solution of NaCl (0.17 M) in methanoland 66.6% heptane. After 2 h at 80 �C this mixture ex-

tracts about 80% of the TAG from sunflower seeds and

about 50, 97, and 99% from soybean seeds, olive fruits,

and acorn fruits, respectively. Even under incomplete

TAG extraction the TAG composition is representative

of the total TAG found in the tissue. All these samples

could be analyzed for TAG composition by GLC. The

remaining TAG-containing heptane solution could beused to get the FAMEs, following a modification of the

method of Garc�ees et al. [13], allowing the analysis of

FAMEs from exactly the same tissue sample.

This method could be very useful to select new vari-

eties of some plants, taking into account the TAG and/

or FA composition of seeds or fruits. It allows one tocarry out the analysis with one-third of the cotyledon,

leaving the rest of the seed containing the embryo. This

fragment could be grown and the new generation col-

lected to continue the selection of any new putative

character.

Acknowledgments

Thanks are due to M.C. Ruiz and D. Cabrera for

skilful technical assistance. We are especially grateful to

A.M. Muro-Pastor for critically reading the manuscript.This work was supported by MCYT Spanish govern-

ment, Junta de Andaluc�ııa, and Advanta Seeds B.V.

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