Monounsaturated Fatty Acid Ether Oligomers Formed during Heating

Article pubs.acs.org/JAFC

Monounsaturated Fatty Acid Ether Oligomers Formed during Heating of Virgin Olive Oil Show Agglutination Activity against Human Red Blood Cells Ioannis S. Patrikios*,†,§ and Thomas M. Mavromoustakos#,⊥ †

School of Medicine, European University Cyprus, Nicosia, Cyprus The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus # National and Kapodistrian University of Athens, Athens, Greece ⊥ National Hellenic Research Foundation, Institute of Biology, Pharmaceutical Chemistry and Biotechnology, Athens, Greece §

ABSTRACT: The present work focuses on the characterization of molecules formed when virgin olive oil is heated at 130 °C for 24 h open in air, which are found to be strong agglutinins. The hemagglutinating activity of the newly formed molecule isolated from the heated virgin olive oil sample was estimated against human red blood cells (RBCs). Dimers and polymers (high molecular weight molecules) were identified through thin layer chromatography (TLC) of the oil mixture. 1H and 13C nuclear magnetic resonance (NMR) and gas chromatography−mass spectroscopy (GC-MS) were the methods used for structural characterization. Among others, oligomerization of at least two monounsaturated fatty acids (FA) by an ether linkage between the hydrocarbon chains is involved. Light microscopy was used to characterize and visualize the agglutination process. Agglutination without fusion or lysis was observed. It was concluded that the heating of virgin olive oil open in air, among other effects, produces oligomerization as well as polymerization of unsaturated FA, possibly of monohydroxy, monounsaturated FA that is associated with strong hemagglutinating activity against human RBCs. The nutritional value and the effects on human health of such oligomers are not discussed in the literature and remain to be investigated. KEYWORDS: hemagglutinins, lectins, fatty acid dimers, vegetable oil processing, virgin olive oil

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INTRODUCTION Oxidation of unsaturated lipids not only produces distasteful odors and flavors but can also affect the nutritional quality and safety by the formation of secondary products in foodstuffs after processing and cooking. Considerable attention has been given to the evaluation and assessment of oxidative and flavor deterioration of fatty acids/lipids as well as the mechanistic concepts of oxidation, aiming to control lipid deterioration.1 Mixtures of free unsaturated long-chain fatty acids (FA) such as those obtained from vegetable oils (i.e., soybean oil) can be polymerized when heated in the presence of water or acidactivated mineral clay, producing mixtures of dimer and trimer acids.2 Dimer acids are unique molecules, and no other commercial chemicals have the same properties. They have a comparatively high molecular weight and, additionally, they contain double bonds that are difficult to conjugate with either the carboxylic group or with one another. These double bonds are reactive toward oxygen and sulfur under many conditions.3 There are several different known methods to dimerize/ oligomerize unsaturated FA including thermal and clay- and peroxide-catalyzed methods.4 Oligomers (dimers C36, trimers C54, and oligomers) are used commercially in many different applications, including resins, corrosion inhibitors, varnishes, and oil additives. Straight-chain FA and their alkyl esters, through a selfcondensation reaction, usually form high molecular weight diand polybasic acids. Two molecules of an unsaturated FA will react with each other to form a dicarboxylic acid with double the original molecular weight. Commercial oligomer acids are mostly © XXXX American Chemical Society

mixtures of 36-carbon dibasic acids, smaller amounts of 54carbon tribasic acids, and trace levels of monomer and higher molecular weight polybasic acids.5 The thermal polymerization of oleic acid (OA), probably by a free radical mechanism, preferentially yields the acyclic form.6−9 Autoxidation of unsaturated fatty acids is another strong possibility when they are excessively heated open in air. Oxygen reacts with many organic substrates to yield hydroperoxides and other oxygenated compounds. This oxidation is a free radical chain reaction with initiation, propagation, and termination processes. Free radicals may be produced by direct thermal dissociation (thermolysis), by hydroperoxide decomposition, by metal catalysis, and by exposure to light (photolysis). Autoxidation of organic substrates depends mostly on their ease in donating hydrogen. With unsaturated fats, susceptibility to autoxidation is dependent on the reactions of available allylic hydrogens with peroxy radicals.1 ROO• + RCH 2CHCHR′ → R•CHCHCHR′ + ROOH

Reaction of oxygen occurs at end carbon positions of the allylic system to produce a mixture of isomeric hydroperoxides. The Received: August 26, 2013 Revised: January 10, 2014 Accepted: January 11, 2014

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dx.doi.org/10.1021/jf403745x | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

previously discussed by Patrikios et al.9 The plates were read at half-hour intervals by observing the bottoms of the wells. The first row of wells served as control. Hemagglutination activity was recognized as the settling of cells in contrast to flowing in the control wells. Titer is expressed as the highest dilution of test samples that still gives agglutination. Specific titer is defined as titer per milligram lipid per milliliter. Thin-Layer Chromatography. Silica gel G plates (20 × 20 cm) (Macherey-Nagel, GmbH & Co., Germany) were prewashed in the developing solvent system (heptane/diethyl ether/acetic acid, 55:45:1, v/v/v) and activated by heating for 1 h at 120 °C under vacuum (15 mm). Sample (20−30 μg, 0.5 mg/mL in chloroform/methanol (1: 1 v/v)) was spotted onto silica gel plates using 2 mL Microcap pipets (Blaubrand, Germany). The plates were dried using a hand-dryer (cool setting) for 5 min before development. The chromatography chamber was saturated with solvent system vapor for 30 min before development of plates. The plates were developed until the solvent front was about 2 cm from the top, removed and air-dried for 30 min, and visualized in iodine vapor. For sample extraction from the plates the bands were visualized by a strip exposed to iodine. Each band was scraped off and eluted with chloroform/methanol (1:1 v/v).11 Heated Extra Virgin Olive Oil Preparation. Virgin olive oil was heated at 130 °C (an average cooking temperature) for 24 h in a metal heating block (USA/Scientific, Olala, FL, USA) in glass tubes open to air. Standard oleic (OA) and ricinoleic acid (RA) (Sigma) were also treated as above. Preparation of Extra Virgin Olive Oil Dispersions. Dispersions were prepared by diluting the samples to 0.5 mg/mL PBS-N. The lipid extract dispersions were filtered through LC PVDF Acrodisc membranes (0.2 μm, Gelman) to remove particulate matter.

termination process can involve combination reactions of the resulting free radicals including production of ether dimers. Oleic acid (OA), a monounsaturated 18-carbon fatty acid, is the major ingredient of virgin olive oil or any other type of olive oil, and a free radical mechanism for thermal dimerization/ oligomerization is very plausible when OA is the starting material. An example of a possible autoxidation mechanism is shown below, with an ether linkaged dimer (PM1) as an end product.

Table 1. Rf Values of the Components of OA, RA, and Virgin Olive Oil, Unheated and Heated (24 h at 130 °C), on Silica Gel TLC Plates in Isooctane/Isopropyl Alcohol/Acetic Acid (95:5:1, v/v/v) Rf values of componentsa item

monomer

OA heated OA

dimer

trimer

0.52 0.87c

0 0 2.3 × 103 7.3 × 102

0.30 0.28 0.26

RA heated RA

Many different structural isomers are possible as a result of head-to-head or head-to-tail alignment of the starting material, as well as many possible positional and geometrical isomers of the double bond.10 It has previously been shown that virgin olive oil becomes a strong hemagglutinin against human red blood cells when heated open in air. The dimer of oleic acid has been characterized as one of the major molecules responsible for the effect.7,9 In this paper an effort is made to investigate if any other byproduct molecules, formed as a result of the virgin olive oil heating process, are associated with hemagglutination abilities and to characterize them structurally.

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0.18

1.0 × 104

0.17

0 0 1.2 × 102 7.2 × 102 1.0 × 103

0.19

0 0 7.5 × 102 7.3 × 102 1.0 × 103

0.62 0.92 0.33 0.24

virgin olive oil heated virgin olive oil

specific activityb

0.65 0.65 0.31 0.25

a Identification of the components of the fractions was by comparison with Rf values of the components of standard commercial monomer, linear dimer, and trimer (Emery). bThe concentration was approximately 0.5 mg/mL. Specific titer is defined as titer per mg lipid per mL. cThe Rf value of the heated OA monomer differs from the Rf value of the nonheated OA monomer because the molecular structure is affected during heating at 130 °C for 24 h (mostly at the double-bond position in the fatty acid chain), probably due to formation of positional and/or geometrical isomers.

MATERIALS AND METHODS

Materials. HPLC grade solvents and fatty acids were obtained from Sigma (St. Louis, MO, USA). The lipid standards were obtained from Larodan Fine Chemicals (Malmo, Sweden). Hemagglutination Assays. Human red blood cells (RBCs) were washed twice with 1 mM phosphate-buffered saline (0.85%)−0.01% sodium azide (pH 7.4). The hemagglutination assays were performed as B



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Figure 1. 1H NMR spectrum of virgin olive oil sample heated at 130 °C for 24 h open in air and esterified with CH2N2. The ester was obtained in CDCl3 solvent at ambient temperature using an INOVA Varian 600 MHz spectrometer.

Figure 2. 1H NMR spectrum of refined virgin olive oil in CDCl3 solvent. The spectrum was obtained at ambient temperature using a Bruker 300 MHz spectrometer. Synthesis of Fatty Acid Methyl Esters. The methyl esters were prepared according to a standard protocol.9 Light Microscopy. A Cytovision Analyzer (Applied Image Co) was used for photographs, attached to a microscope (Zeiss Axioskop). Agglutinated cells were taken from microtiter plates that were titered and allowed to develop for approximately 30 min. The following preparations were used: (1) untreated human RBCs; (2) active lipid extract from heated virgin olive oil (1 mg/mL); (3) unheated virgin olive

oil preparations (1 mg/mL). All samples were dissolved in PBS-N. Fresh human RBCs were washed once with citrate because the cells retain their shape much better in citrate than in PBS and, therefore, allow better photographs to be taken. It should be noted that cells washed in citrate will maintain their integrity for only approximately 2 h. Nuclear Magnetic Resonance Spectroscopy. CDCl3 (99.5%) ampules were purchased from Merck (Darmstadt, Germany) and ultraprecision NMR tubes from Peypin (France) and Wilmad 535-5 mm C



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Table 2. 1 H NMR Chemical Shifts of Virgin Olive Oil in CDCl3 Solvent peak

δ

proton

assignment

1 2 3 4 5 6 7 8 9 10 11 12

8.45 7.27 5.29 5.15 4.19 2.76 2.2 2.02 1.6 1.2 0.95 0.85

pyrazine CHCl3 CHCH CHOCOR CH2OCOR CHCHCH2CHCH CH2COOH CH2CHCH CH2CH2COOH (CH2)n CH2CH2CH2CH3 CHCHCH2CH3

all unsaturated fatty acids glycerol (triacylglycerols) glycerol (triacylglycerols) linoleyl and linolenyl all acyl chains all unsaturated fatty acids all acyl chains all acyl chains linolenyl all acids except linolenyl

Mass Spectrometry. Mass spectra were obtained with a TSQ 7000 Finnigan MAT spectrometer using electron spin ionization (ESI) and spray voltage (4.5 kV). The sheath gas pressure (N2) during the experiment was 35 psi and the capillary temperature, 200 °C. Samples were dissolved in a mixture of CH3OH/CHCl3.

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RESULTS AND DISCUSSION The approach by which oils and foodstuffs in general are manufactured, processed, cooked, and used affects their nutritional value and safety. Many studies in the literature have discussed the oxidation products of olive oil. In 2002 Patrikios and in 2003 Patrikios et al. reported the hemaglutinating properties of oxidation and thermal degradation products of lipid extracts isolated from heat-processed foodstuffs including virgin olive oil.8,9 In the present study we focused on the molecular structure of another possible specific molecule that is possibly formed in the virgin olive oils under the above conditions and which promotes hemagglutinating activity with human RBCs. Thin-layer chromatography on silica gel, in a system that discriminates by the number of carboxyl groups (free fatty acids are released from glycerol after the sample heating preparation process), was used to resolve the components of the heated virgin olive oil (Table 1). Bands were cut out, eluted, and titered against human RBCs. The identification of the components of the fraction was carried out by comparison with Rf values of the components of a heated sample of oleic acid, ricinoleic acid (RA), and standard commercial monomer, dimer and trimer (Emery). A portion of byproducts in the heated virgin olive oil sample showed the same Rf values as the corresponding Rf values of the monomeric, oligomeric, and polymeric byproducts of the heated sample of OA, RA, and the commercial oligomers. As reported by Patrikios, this might be the result of hydrolysis, which can take place under heat processing in air, of the triacylglycerols,

(Spintec-Rototec). The high-resolution spectra in CDCl3 were obtained using Bruker 300 AC and Varian INOVA 600 instruments. All data were collected using pulse sequences and phase cycling routines provided in the Bruker and Varian libraries of pulse programs. 1H NMR spectra in the Bruker instrument were recorded using the following acquisition parameters: pulse width (PW), 3.0 μs; spectral width (SW), 2513 Hz; data size (TD), 32K; recycling delay (RD), 1.0 s; number of transients (NS), 16; and digital resolution, 0.076 Hz pt−1. DQF-COSY and 1 H−13C HSQC spectra experiments were performed with gradients at 600 MHz. The 1H sweep width was 9820 at 600 MHz. Typically homonuclear proton spectra were acquired with 4096 data points in t2, 16−64 scans, 256−512 complex points in t1, and relaxation delay of 1−1.5 s. Data were processed and analyzed with the v-NMR software package from Varian. Spectra were zero-filled two times and apodized with a square sine-bell function shifted by π/2 in both dimensions. The 1 H−13C HSQC spectrum was recorded with 1024 data points in t2, 16 scans per increment, 128 complex points in t1, and a relaxation delay of 1 s. The 13C spectral width was 20000 Hz.

Figure 3. 2D NMR spectrum of HSQC virgin oil sample heated at 130 °C for 24 h open in air and esterified with CH2N2. The ester was obtained in CDCl3 solvent at ambient temperature using an INOVA Varian 600 MHz. D



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Figure 4. 13C NMR spectrum of virgin olive oil in CDCl3 solvent. The spectrum was obtained at ambient temperature using a Bruker 300 MHz spectrometer.

Table 3. 13 C NMR Chemical Shifts of Virgin Olive Oil in CDCl3 Solvent peak

δ

carbon

compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

173.26 172.81 129.98 129.67 128.06 127.86 77.01 68.92 62.18 34.18 34.02 31.88 29.1−29.8 27.16 25.81 24.84 22.65 14.15 0

C-1, sn-1,3 C-1, sn-2 C-10 C-9 C-10 C-12 CDCl3 CHO−, sn-2 CH2O−, sn-1,3 C-2, sn-2 C-2, sn-1,3 ω3 (CH2)n allylic: C-8−C-11 oleyl, C-8−C-14 linoleyl diallylic: C-11 linoleyl, C-11−C-14 linolenyl C-3 ω2 ω1 (−CH3) TMS

triacylglycerols triacylglycerols oleyl oleyl linoleyl linoleyl solvent triacylglycerols triacylglycerols all acyl chains all acyl chains saturated, n-9 and n-6 acids all acyl chains

all acyl chains all acyl chains all acyl chains

polymerization.9 The TLC shows two spots with slightly different Rf values for dimers. One of the dimer molecules with

releasing oleic acid, which after free radical formation and oxidation can result in dimerization, oligomerization, and E



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Figure 5. Light micrographs of human erythrocytes in citrate buffer (pH 7.4): (a) with unheated virgin olive oil; (b) with the isolated virgin olive oil oligomer preparation (1.0 mg/mL).

A spot with about the same Rf is observed when commercial RA was treated under the same conditions (Table 1). The 1H NMR spectrum of the compound is shown in Figure 1. To characterize it, several 1H NMR spectra from virgin olive oil samples originated from different countries were obtained. A representative one is shown in Figure 2. The sample was run in CDCl3 and using a known amount of pyrazine in case a

Rf 0.31 was characterized by Patrikos et al. as a linear dimer of oleic acid (dioleyl), dimerized by −C−C− linkage.9 This dimer was shown to be associated with strong hemagglutinating properties against human RBCs. The second dimer formed with Rf 0.25 is now characterized as an ether-linked unsaturated dimer and is also shown to be associated with hemagglutinating properties against human RBC. F



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Figure 6. MS spectrum of virgin olive oil sample heated at 130 °C for 24 h open in air and esterified with CH2N2. The MS spectrum was obtained using a TSQ 7000 Finnigan MAT spectrometer and electron spin ionization with spray voltage (4.5 kV).

Table 4. Major Mass Spectrum (MS) Peaks (m/e) and Their Interpretation peak m/e

interpretation

637.5

the ether dimer has an m/e 606 the sample was dissolved in CH3OH/CHCl3 CH3OH has an m/e 32 606 + 32 = 638

549.5

2[CH3COH]+ = 44 × 2 = 88 637.5 − 88 = 549.5

505.5

[CH3COH]+ = 44 549.5 − 44 = 505.5

461.3

[CH3COH]+ = 44 505.5 − 44 = 461.5

417.2

[CH3COH]+ = 44 461.3 − 44 = 417.3

oils are lacking peaks in the region of 50 ppm (Figure 4 and Table 3), in agreement with data obtained by Sacchi.14 The 2D COSY spectrum showed a correlation between the assigned peaks as CH−O−CH with CH2 as expected. Panels a and b of Figure 5 show human RBCs that were mixed with unheated virgin olive oil and with the isolated oligomer preparation, respectively. As seen in Figure 5b, mixtures of chains and rosettes were observed by light microscopy (LM), but no lysis or fusion was apparent. Agglutination was found to involve cell clumping and to be time-dependent. The agglutination activity, which we observed, most likely is due to a partial insertion of the dimer/oligomer formed into red cell membrane in a way that links red cell without affecting membrane dynamics and permeability properties. ESI mass spectroscopy of the oligomer isolated from the heated sample of virgin olive oil after methylation suggests the possible existence of the proposed dimer, an acyclic “hydro” dimer that contains two cis and/or trans double bonds and ether linkages at the 8, 9, 10, and 11 carbons between two aliphatic (hydrocarbon) chains. The interpretations of all major peaks in the Figure 6 mass spectrum are shown in Table 4. In particular, the peak at 637.5 has a molecular weight very close to that of PM1 + CH3OH (CH3OH was used as a dilution solvent along with CHCl3). The peak at 549.5 is 88 units smaller and may correspond to a dimer without two positively charged ions of m/e 88 2[CH3COH]+. Interestingly, the most intense peaks observed in the spectrum differ from the ones with higher m/e by 44.

quantitative result was necessary. This method was developed in two previous publications by Mavromoustakos et al.12,13 As can be observed in the representative spectrum shown in Figure 2, the peaks are almost identical with those reported by Sacchi et al. and are shown in Table 2.14 All olive oils analyzed were missing the peaks in the region of 3.5−3.7 ppm observed for the sample under investigation. We made an effort to characterize these peaks. First, we have drawn several plausible structures and used NMR databases to simulate the spectrum. A compound that gave a 1H NMR spectrum similar to the experimental was PM2 (esterified). The peak at 3.5 ppm corresponds to a methoxy group, whereas the peak at 3.65−3.7 ppm corresponds to CH−O−CH. HSQC experiment is in agreement with this assignment. C−H correlation between the two critical peaks at the region of 3.5−3.7 ppm and the corresponding peaks centered at 50 ppm were observed in HSQC spectrum (Figure 3). 13C NMR spectra of virgin olive G



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(11) Kates, M. Techniques in Lipidology; North Holland/American Elsevier: New York, 1986. (12) Mavromoustakos, T.; Zervou, M.; Theodoropoulou, E.; Panagiotopoulos, D.; Bonas, G.; Day, M.; Helmis, A. 13C NMR analysis of the triacylglycerol composition of Greek virgin olive oils. Magn. Reson. Chem. 1997, 35, S3−S7. (13) Mavromoustakos, T.; Zervou, M.; Bonas, G.; Kolocouris, A.; Petrakis, P. A. Novel analytical method to detect adulteration of virgin olive oil by other oils. J. Am. Oil. Chem. Soc. 2000, 77 (4), 405−411. (14) Sacchi, R.; Addeo, F.; Paolillo, L. 1H and 13C NMR of virgin olive oil. An overview. Magn. Reson. Chem. 1997, 35, S1333−S145.

We concluded that virgin olive oil can undergo oligomerization through free radical mechanism of the oleic acid and autoxidation when heated at 130 °C for 24 h open in air. Subsequently, dioleyl or oligomers are formed by a direct linkage of the accumulated free radicals or by an intermediated peroxy radical formation (through autoxidation reaction).7 Thus, when virgin olive oil is consistently heated open in air it can possibly undergo nutritional damage with a possible direct but as yet unknown effect on human health.

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AUTHOR INFORMATION

Corresponding Author

*(I.S.P.) Mail: School of Medicine, European University Cyprus, 6 Diogenes Street, 2404 Engomi, P.O. Box 22006, 1516 Nicosia, Cyprus. Phone: 00-357-99097856. E-mail: [email protected]. Author Contributions

Both authors interpreted the data. I.S.P. drafted the report tables and figures, and all authors critically revised and approved the final version. I.S.P. was responsible for the laboratory experiments, and T.M. was responsible for the NMR and GC-MS experiments. Both I.S.P. and T.M. performed the literature search, and both contributed to the structural elucidation of the active molecule and the rationale of the involved biochemical mechanisms. Both authors vouch for the accuracy and completeness of the data, and both authors read and approved the final manuscript. Funding

This work was supported by a grant from the Research Promotion Foundation of Cyprus, RPF 06/99. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Cyprus Institute of Neurology and Genetics for hosting the project. We also acknowledge E. Siapi for obtaining MS spectrum and Simona Golic Grdadolnik, who obtained some of NMR spectra. We especially thank Elsa Tsartsidou for her contribution in the preparation of the tables.

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REFERENCES

(1) Frankel, E. N. Lipid oxidation. Prog. Lipid Res. 1980, 19, 1−22. (2) Conroy, N. H. Polymerization of unsaturated fatty acids. U.S. Patent 3,632,822, 1969. (3) Cowan, J. C. Dimer acids. J. Am. Oil Chem. Soc. 1962, 39, 534−545. (4) Leonard, E. C. Dimer acids. In Kirk-Othmer: Encyclopedia of Chemical Technology, 3rd ed.; Wiley Interscience: Hoboken, NJ, USA, 1979; pp 768−782. (5) Leonard, E. C. The Dimer Acids; Hunko Sheffield Chemical: Memphis, TN, USA, 1975; pp 1−112. (6) Myers, L. D.; Goebel, C. G.; Barrett, F. O. Process for polymerizing unsaturated fatty acids. U.S. Patent 3,076,003, 1960. (7) Patrikios, I. S.; Britton, O’N.; Bing, D. K.; Russell, C. S. Heating unsaturated fatty acids in air produces hemagglutinins. Biochim. Biophys. Acta 1994, 1212, 225−234. (8) Patrikios, I. S. Lipid extracts isolated from heat processed food show a strong agglutinating activity against human red blood cells. Food Res. Int. 2002, 35 (6), 535−540. (9) Patrikios, I. S.; Patsalis, P. C. Monounsaturaded fatty acid oligomerization is responsible for the agglutination activity of heated virgin olive oil. Food Res. Int. 2003, 36, 480−489. (10) McMahon, D. H.; Crowell, E. P. Characterization of products from clay-catalyzed polymerization of tall oil fatty acids. J. Am. Oil Chem. Soc. 1974, 51, 522−527. H


Monounsaturated Fatty Acid Ether Oligomers Formed during Heating

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