A Teaching Laboratory for Comprehensive Lipid Characterization from

Laboratory Experiment pubs.acs.org/jchemeduc

A Teaching Laboratory for Comprehensive Lipid Characterization from Food Samples Kestutis Bendinskas,*,† Benjamin Weber,† Tamara Nsouli,† Hoangvy V. Nguyen,† Carolyn Joyce,† Vadoud Niri,† and Thorsten W. Jaskolla‡,§ †

Department of Chemistry, State University of New York at Oswego, Oswego, New York 13126, United States Institute of Hygiene, University of Münster, Robert-Koch-Strasse 41, 48149 Münster, Germany § Dr. Franz Köhler Chemie GmbH, Werner-von-Siemens-Strasse 22-28, 64625 Bensheim, Germany ‡

S Supporting Information *

ABSTRACT: Traditional and state-of-the-art techniques were combined to probe for various lipid classes from egg yolk and avocado qualitatively and quantitatively. A total lipid extract was isolated using liquid−liquid extraction. An aliquot of the total lipid extract was subjected to transesterification to form volatile fatty acid methyl esters suitable for gas chromatography to quantify fatty acyl residues in both food samples. Another total lipid aliquot was separated into two lipid fractions of higher and lower polarity using silicic acid column chromatography. All fractions were analyzed using thin-layer chromatography and were checked for cholesterol using the Liebermann-Burchard test. In addition, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry using 2,5-dihydroxybenzoic acid and 4-chloro-α-cyanocinnamic acid matrixes was performed to analyze the composition of separated lipid fractions. Egg yolk exhibited phosphatidylcholines and phosphatidylethanolamines in the more polar fraction and cholesterol in the less polar one. Avocado contained detectable amounts of the nonpolar triglyceride triolein. The experiments can be split between three 3-h laboratory periods to allow upper-division students to gain exposure to both lipid theory and hands-on experimental lipid analysis. KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Biochemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Testing/Assessment, Lipids, Mass Spectrometry, Bioanalytical Chemistry, Natural Products

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INTRODUCTION Lipids constitute one of the four major classes of biological molecules and are involved in a vast array of intra- and extracellular functions. It is of great interest within the scientific community to be able to extract and categorize lipids from different sources. To analyze the lipids present in these food products, a total lipid extract was obtained. For isolation, a modified Bligh & Dyer, and Folch liquid−liquid extraction was used.1 A suggested modification to the original method was to replace chloroform, which should be avoided in a teaching laboratory, by less toxic dichloromethane (DCM).2 A portion of the total extract was then separated into two fractions of higher and lower hydrophobicity using silicic acid column chromatography. With a pre-column and two post-column fractions, various analyses were carried out to determine the types of lipids present in the food sources. The total lipid extract was subjected to transesterification to yield fatty acid methyl esters (FAMEs) suitable for gas chromatography (GC) or GC coupled to mass spectrometry (GC/MS) analysis.3 Thin-layer chromatography (TLC) followed by various visualization techniques was used to test the fractions of different hydrophobicity for the presence of lipids known to be © XXXX American Chemical Society and Division of Chemical Education, Inc.

commonly found in egg yolk and avocado. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was then used to determine components of the two separated lipid fractions.4 For the experiment, students used 2,5-dihydroxybenzoic acid (DHB) matrix for the less hydrophobic lipid fraction and 4-chloro-α-cyanocinnamic acid (ClCCA) matrix for the more hydrophobic one. Lastly, all three fractions were tested for cholesterol using the qualitative Liebermann-Burchard test.5 Finally, students compared their results with the reference data found on the National Nutrient Database for Standard Reference6 and discovered that these food sources contain very different lipid species. The experiment was performed in biochemistry teaching laboratories for the last six years. The GC/MS and MALDI parts of the experiment were introduced two years ago. Thirtytwo students performed the complete laboratory experiment. GC/MS and MALDI-TOF experiments with lipids have been published earlier in this Journal.3,4 However, the presented experiment is significantly more comprehensive. It includes lipid fractionation and juxtaposes a variety of qualitative and quantitative traditional and modern techniques of lipid analysis

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dx.doi.org/10.1021/ed400586z | J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

and demonstrates how they supplement and complement each other. It may be considered to be an introductory laboratory experiment to modern lipidomics. This laboratory experiment is designed as a starting point for upper-division undergraduates to understand how to carry out a comprehensive lipid analysis in different sources of food, such as egg and avocado.

Table 1. Percent Lipid Composition (w/w) for Both Analyzed Food Sourcesa Class Average Data (N = 18) Student Research Data (N = 6) Reference Value6

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Egg yolk

Avocado

26 ± 12% 20.1 ± 1.4% 26.6%

17.6 ± 6.6% 12.2 ± 0.4% 14.7%

From all trials, the averaged values ± standard deviations were calculated. a

METHODS AND PROCEDURES This experiment analyzes egg yolk and Hass avocado (i.e., typical avocado) lipid compositions. Both samples were purchased from a local produce store. All reagents and supplies were from Thermo Fisher Scientific, Waltham, MA, unless otherwise specified. FAMEs were analyzed using gas chromatography with flame ionization detector (GC-FID). The comprehensive procedure is described in full detail in the Supporting Information. In schools that lack GC/MS and MALDI-TOF instruments, high quality data can be provided to students from the Supporting Information, while students can perform all remaining experiments. Students work in twoperson teams with each partner analyzing a different food source. Both students analyze both sets of data in their reports. Students are encouraged to bring an additional food source to analyze if they wish to do so.

Using the total lipid extract weight, the percent lipid composition (w/w) of each food sample was calculated (Table 1). The percentages obtained by students indicated that the applied lipid extraction procedure was not quantitative, as expected. Transesterification was performed on a portion of the total lipid extract to yield a mixture of FAMEs, which were determined to be about 40% (w/w) for egg yolk and about 30% (w/w) for avocado by means of their weight. Standard FAMEs and samples were analyzed using GC-FID and identified by means of their retention times (Table 2). Egg Table 2. Fatty Acyl Residues Identified as Fatty Acid Methyl Esters Using GC-FIDa

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HAZARDS Precautions should be taken for the following listed materials. Dichloromethane, methanol, and toluene are flammable and toxic. Hexane is flammable, neurotoxic, and irritant. Sulfuric acid is very corrosive and toxic. Acetic acid and acetic anhydride are corrosive and toxic. Ninhydrin spray is toxic. Iodine is toxic and volatile. Iron(III) chloride spray is very corrosive. Acetonitrile is toxic upon ingestion in significant amounts. The following solids are used in relatively small amounts or in solutions and do not pose significant risks to experimenters: 2,5-dihydroxybenzoic acid, 4-chloro-α-cyanocinnamic acid, silicic acid, calcium chloride, magnesium chloride, sodium chloride, sodium sulfate, and sodium bicarbonate. All food samples should be handled with gloves or, alternatively, hands should be carefully washed afterward. All volatile solvents should be handled in a hood.

Fatty acyl residue Myristoyl (14:0) Palmitoleyl (16:1) Palmitoyl (16:0) Oleoyl/Linoleoyl (18:1/18:2) Stearoyl (18:0) Arachidonyl (20:0) Palmitoleyl (16:1) Palmitoyl (16:0) Oleoyl/Linoleoyl (18:1/18:2) Arachidonyl (20:0)

Experimental relative abundance (%) Egg Yolk 0.7 1.9 36.8 45.6 10.4 4.6 Avocado 4.2 28.4 63.8 3.6

Reference6 relative abundance (%)

Retention time (min)

1.0 3.2 26.1 58.8 8.9 2.0

4.6 6.4 6.6 8.2 8.5 9.6

6.2 23.3 69.6 0.3

6.4 6.6 8.2 9.6

a Relative abundances (%w/w) were calculated by the instrumentgenerated peak areas.

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RESULTS AND DISCUSSION In short, a total lipid extract was isolated using liquid−liquid extraction. An aliquot of the total lipid extract was subjected to transesterification to form volatile fatty acid methyl esters suitable for gas chromatography to quantify fatty acyl residues in both food samples. Another total lipid aliquot was separated into two lipid fractions of higher and lower polarity using silicic acid column chromatography. All fractions were analyzed using TLC and were checked for cholesterol using the LiebermannBurchard test. In addition, MALDI-TOF MS was performed to analyze the composition of separated lipid fractions. The data presented in this paper and Supporting Information represent averages of three duplicate runs done by an experienced undergraduate student and averages of class data for the most recent laboratory experiment performed by 18 students. Since it is challenging to extract lipids quantitatively,4b,7 class data show significantly larger standard deviations compared to literature values (Table 1). A complete set of experimental data are provided in instructor’s manual. All MS spectra provided represent typical class data.

yolk from the batch of eggs investigated consisted of 52.5% saturated (literature value: 38%6) and 47.5% unsaturated (literature value: 62%6) FAMEs. The avocado contained 32% saturated (literature value, 24%6) and 68% unsaturated (literature value, 76%6) FAMEs. GC/MS identified and quantified the species given in Table 2. Oleic acid methyl ester and linoleic acid methyl ester eluted almost simultaneously at the selected GC parameters on a 30 m GC column; the separation can be improved using longer columns.3a TLC analysis was used to identify more polar (Table 3) and less polar (Table 4) common lipids found in both investigated food sources. The more polar egg yolk fraction contained various phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) (Table 3) in agreement with Bender et al.3a In the less polar egg yolk fraction (Table 4), a spot with the same Rf as a cholesterol standard was detected and was confirmed to be cholesterol using iron(III) chloride spray. Squalene was used in this experiment to emphasize the color change of cholesterol derivatives during the ferric chloride test. Squalene as a polyisoprenoid does not exhibit a phenolic structure, but shows B




detectable in the less polar egg yolk fraction, but not in either avocado lipid extract fraction. MALDI-TOF MS was used to analyze the two lipid fractions of different polarity from both food samples. Figure 1S (Supporting Information) shows the mass spectrum generated from the more polar egg yolk fraction. Table 6 gives a list of all

Table 3. TLC Analysis of the Egg Yolk and Avocado Lipid Extract Fractions of Higher Polaritya Rf

Standard Phosphatidylcholines Phosphatidylethanolamines Phosphatidylcholines Phosphatidylethanolamines

Primary or secondary amine

Egg Yolk 0.15 0.90 Avocado 0.15 0.90

Detected in sample

No Yes

Yes Yes

No Yes

No No

Table 6. Identified and assumed lipids of the more polar egg yolk lipid fraction using MALDI-TOF MS with a mass accuracy of ±0.04 Da

a

The Rf values and ninhydrin spray results (as a probe for primary and secondary amines) correspond to the standards. Dichloromethane/ methanol/acetic acid/water (100:23:5:1 (all v/v)) was used as mobile phase.

Table 4. TLC Analysis of the Egg Yolk and Avocado Lipid Extract Fractions of Lower Polaritya Rf

Standard Cholesterol Squalene Triolein

0.51 0.94 0.88

Cholesterol Squalene Triolein

0.51 0.94 0.88

UV active at 254 nm

Reaction with FeCl3

Detected in sample

Yes Yes No

Yes No No

Yes Yes No

No No Yes

Egg Yolk Yes Yes Yes Avocado Yes Yes Yes

a

The Rf values, UV lamp results (detection of unsaturated lipids), and iron(III) chloride spray results (detection of cholesterol as a phenolic structure and detection of squalene via charring) correspond to the standards. Hexane/diethyl ether/acetic acid (49:29:5 (all v/v)) was used as mobile phase.

a color change upon treatment with FeCl3, which most probably is due to charring.8 In the more polar avocado fraction (Table 3), no sample spots matched standard spots, whereas the less polar avocado sample (Table 4) was found to contain only triolein, a triacylglyceride with three oleoyl residues, in agreement with the previously discussed GC data and with Bender et al.3a The Liebermann-Burchard test for unsaturated steroids (e.g., cholesterol) was performed for both egg yolk and avocado (Table 5). A negative control yielded a clear solution, while a positive control turned a green/blue color. For egg yolk, it was found that an unsaturated steroid (which was identified to be cholesterol by other tests) was present in the less but not in the more polar lipid fraction, indicating a successful separation of less and more polar lipids by silicic acid chromatography. This also confirmed the TLC experiments by which cholesterol was

Color

Unsaturated steroids

Positive control Negative control More polar egg yolk fraction Less polar egg yolk fraction Total lipids egg yolk fraction More polar avocado fraction Less polar avocado fraction Total lipids avocado fraction

Blue/Green Colorless Yellow Dark blue/green Green Very light green Light green/purple Light green/purple

Yes No No Yes Yes No No No

Calculated monoisotopic peak of protonated species (m/z)

718.53 740.51

718.54 740.52

C39H77NO8P C41H75NO8P

744.54

744.55

C41H79NO8P

746.57 758.58

746.57 758.57


760.61 768.57 782.59

760.59 768.55 782.57

786.63

786.60

C42H83NO8P C43H79NO8P C42H82NO8PNa C44H81NO8P C44H85NO8P

788.66

788.62

C44H87NO8P

790.61 806.56

790.63 806.57


808.60

808.59

C46H83NO8P

810.64

810.60

C46H85NO8P

820.55

820.59

C47H83NO8P

822.59

822.60

C47H85NO8P

824.60

824.62

C47H87NO8P

Assumed lipid composition PE 18:0/16:1 PE 16:0/20:4 PE 18:2/18:2 PE 18:1/18:1 PE 18:0/18:2 PE 18:0/18:1 PC 16:0/18:2 PC 16:1/18:1 PC 16:0/18:1 PE 18:0/20:4 PC 16:0/18:1 PC 18:2/18:2 PC 16:0/20:2 PC 18:0/18:2 PC 16:0/20:1 PC 18:0/18:1 PC 18:0/18:0 PC 16:0/22:6 PC 18:2/20:4 PC 16:1/22:4 PC 18:1/20:4 PC 16:0/22:4 PC 18:0/20:4 PE 22:2/20:4 PE 20:2/22:4 PE 20:1/22:4 PE 22:1/20:4 PE 20:0/22:4 PE 20:2/22:2

identified peaks in Supporting Information Figure 1S using reference mass values9 with a mass tolerance of ±0.04 Da (about 50 ppm) for all peaks. Most of the identified peaks were PCs with palmitoyl and oleoyl residues. This finding was confirmed by the GC data that showed that these two fatty acyl residues accounted for 82.4% (w/w) of the egg yolk fatty acids converted to FAMEs. Due to the low overall Na+ content of the lipid fractions, which were initially generated by extracting the food samples with DCM/methanol = 2:1 (v/v), nearly no sodium adducts were generated. If considerable Na+ amounts would be included in the lipid fractions, more or less all detected PC species would occur as protonated as well as sodiated species, which were not observed. Therefore, it was reasonable to assume that the few detected peaks that might result from sodiated lipid species referred, in fact, to protonated lipids. However, in case of m/z 782.57, the possibility of sodiated PC 16:0/18:1 instead of or additionally to protonated PC 18:2/18:2 could not be excluded without further MS/MSfragmentation analysis since the peak of protonated PC 16:0/

Table 5. Results of the Liebermann-Burchard Tests for Unsaturated Steroids Sample

Chemical composition of protonated/ sodiated species

Detected monoisotopic peak (m/z)

C




18:1 at m/z 760.59 was the strongest one in the spectrum, which might also result in low amounts of the corresponding sodiated adduct. For the unsaturated fatty acyl residues, the position(s) of the double bond(s) could not be concluded from the detected mass alone. Nevertheless, for the detected unsaturated fatty acyl residues 18:2 and 20:4, only linoleoyl and arachidonyl residues are known. For the residues with the composition 18:1, there is only one alternative double bond position known in addition to that of oleoyl (double bond at position 9), which is petroselinoyl (double bond at position 6), found only in oils of the Umbelliferae family.9b Referring to mono-unsaturated PC/PE lipid species, the sn-1 position of phospholipids is typically occupied by the saturated fatty acyl residue, whereas the sn-2 position is typically taken by the unsaturated residue. Thus, exact lipid structure elucidation would require MS/MS-analysis to differentiate between phospholipid protonation and sodiation. No lipids from avocado were identified from its more polar lipid extract fraction; the spectrum was simply that of the DHB matrix. This was not a surprise since the TLC data (Table 3) suggested very low abundance of more polar lipids in avocado. Nonpolar lipids were successfully identified using ClCCA as a matrix.10 The mass spectrum of a triolein standard (Supporting Information Figure 2S) showed a distinct peak at m/z 603.5 due to neutral loss of one oleoyl residue (C18:1) after initial triolein protonation with prompt fragmentation11 in addition to sodiated triolein at m/z 907.8. Although there might be generation of additional triolein fragments depending on the selected MS parameters, m/z 603.5 was one of the most prominent ones.12 When the less polar avocado lipid fraction was tested, identical peaks at m/z 603.5 and 907.8 were detected (Supporting Information Figure 3S), which strongly indicated the presence of significant triolein amounts in avocado, confirming the results from GC (Table 2). Two other strong peaks at m/z 578.8 and 636.7 could not be referred to glycerophospholipids or di/triacylglycerols according to the lipid maps database9 and probably corresponded to other triolein fragmentation products or other lipid species. A standard cholesterol mixture was also tested using ClCCA as matrix (Supporting Information Figure 4S). Some peaks for the more polar egg yolk lipid fraction could not be correlated to certain cholesteryl species. The identified and assumed cholesteryl esters (CEs) are listed in Table 7. In comparison to a positive ion mode mass spectrum of the less polar egg yolk lipid fraction (Supporting Information Figure 5S), peaks at the same m/z ratios were detectable at m/z 637.52, 647.53, 663.59, and 685.38, which once again confirmed the presence of cholesterol as corresponding esters

in egg yolk. In addition, further CEs seem to be present in egg yolk (Table 8). Table 8. CEs Assumed to Be Additionally Present in the Less Polar Egg Yolk Lipid Fractiona

Calculated monoisotopic peak (m/z)

Chemical composition of sodiated cholesteryl ester species

637.49 647.59 663.56

637.50 (sodiated) 647.57 (sodiated) 663.57 (sodiated)

C43H66O2Na C43H76O2Na C43H76O3Na

685.56

685.55 (sodiated)

C45H74O3Na

693.56

693.56 (sodiated)

C47H74O2Na

Calculated monoisotopic peak (m/z)

Chemical composition of sodiated cholesteryl ester species

639.51 665.72 687.41

639.51 (sodiated) 665.53 (sodiated) 687.57 (sodiated)

C43H68O2Na C45H70O2Na C45H76O3Na

Assumed lipid composition CE(16:4) CE(18:5) CE(18:2 (OH))

a

These are in addition to those listed in Table 7 using MALDI-TOF MS; a mass accuracy of ±0.2 Da is due to peak overlapping at m/z 665.72 and 687.41.

Numerous lower abundant lipids, including entire groups of lipids, lipid dimers, difficulties in differentiating between lipid isomers and sodium adducts, and use of negative-ion mode MALDI MS analysis are further noted in the Supporting Information. After completing the entire laboratory experiment and writing reports, students filled out a survey reflecting their experience. A full summary of responses is included in the Supporting Information. Overall, students found that their knowledge of various lipid classes and the techniques used in the lab improved after completion of the laboratory (average rating was about 4 out of 5). Students also expressed that this lab was generally more interesting than other laboratories they had performed and agreed that it should be continued in future years.

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CONCLUSIONS This laboratory provided upper-division biochemistry students with a flexible and dynamic comprehensive experiment focusing on lipid extraction, separation, and several analysis techniques. The entire experiment was performed in three 3-h teaching laboratory periods and provided an introduction to lipidomics.

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ASSOCIATED CONTENT

S Supporting Information *

Student manual and instructor notes. This material is available via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

Table 7. Identified and Assumed CEs of the More Polar Egg Yolk Lipid Fraction Using MALDI-TOF MS with a Mass Accuracy of ±0.02 Da Detected monoisotopic peak (m/z)

Detected monoisotopic peak (m/z)

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank SUNY Oswego, specifically the Scholarly and Creative Activity Committee and the Provost office, for their support. We thank Wayne Stripe for developing the original experiment, short of GC/MS and MALDI. We thank David Kiemle, the instructional support specialist at SUNY-ESF, for his help with the identification lipids on the initial set of runs for GC/MS. Financial support by the Deutsche Forschungsgemeinschaft (Grant JA2127/1-1 to T.W.J.) is gratefully acknowledged.

Assumed lipid composition CE(16:5) CE(16:0) CE(16:0 (OH)) CE(18:3 (OH)) CE(20:5) D



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REFERENCES

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A Teaching Laboratory for Comprehensive Lipid Characterization from

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