Chem. Res. Toxicol. 1996, 9, 1001-1006
1001
Products of Aniline and Triglycerides in Oil Samples Associated with the Toxic Oil Syndrome Helen H. Schurz,*,† Robert H. Hill,† Manuel Posada de la Paz,‡ Rossanne M. Philen,† Ignacio Abaitua Borda,‡ Sandra L. Bailey,† and Larry L. Needham† Centers for Disease Control and Prevention (CDC), 4770 Buford Highway NE, Mailstop F17, Atlanta, Georgia 30341-3724, and Direccion General de Ordenacion de la Investigacion y Formacion, Fondo de Investigacion Sanitaria, Ministerio de Sanidad y Consumo, Sinesio Delgado 6, 28029 Madrid, Spain Received October 30, 1995X
The toxic oil syndrome (TOS) was a devastating disease that occurred in Spain in 1981. The disease was associated with the consumption of aniline-denatured and refined rapeseed oil that had been illegally sold as olive oil. Many aniline-derived oil components have been identified in the oils; however, no etiological agent has ever been identified for this disease. We have continued the study of the TOS problem by applying new technology in the form of liquid chromatography interfaced via atmospheric pressure ionization with tandem mass spectrometry. Using liquid chromatography tandem mass spectrometry, we studied diluted TOS-associated oils by direct analysis without prior sample treatment. Using this technology, we found new classes of compounds that are associated with disease-related oils. The compounds that have been identified are esters and ester amides of 3-(N-phenylamino)-1,2propanediol and are products of aniline and triglycerides. Because of the varied fatty acid (oleic acid, etc.) content of the oils, many variations of the above compounds are possible. We now report the identities of more than 20 compounds not previously identified. These compounds are strongly associated with oils that caused the toxic oil syndrome. We believe these compounds should be considered for future animal studies.
Introduction (TOS)1
Since the toxic oil syndrome occurred in 1981, researchers worldwide have pursued the etiological agent of this devastating disease (1-3). At the height of the epidemic, more than 20 000 people in the central and northwestern regions of Spain were afflicted, and of these, 11 000 required hospitalization. More than 800 of the afflicted have died since the epidemic, and it has become difficult to pinpoint the number of deaths that TOS directly contributed to or caused. Initially, TOS exhibited itself as an atypical pneumonia suggesting a bacterial or viral origin. As time passed, however, TOS developed into a severe chronic immunologic disease. Early exhaustive laboratory investigations discounted infectious agents as potential TOS agents. Complicating the diagnosis of TOS was a high eosinophilia count occurring among the victims that suggested a parasitic disease, yet laboratory testing for parasites was negative. Tabuenca (4) was first to recognize an association between TOS and the consumption of a food oil that was being sold in mercadillos (outdoor markets). The Spanish government instituted a massive oilexchange program to remove the tainted oil from the population. All cooking oil, regardless of its origin, could be traded in for pure olive oil. This recall removed most of the tainted oil from the market, and the number of new TOS cases decreased. †
Centers for Disease Control and Prevention (CDC). Ministerio de Sanidad y Consumo, Madrid. Abstract published in Advance ACS Abstracts, August 1, 1996. 1 Abbreviations: TOS, toxic oil syndrome; PAP, 3-(N-phenylamino)1,2-propanediol; LC-MS/MS, liquid chromatography tandem mass spectrometry; DPAP, diester of DPAP; APCI, atmospheric pressure chemical ionization; MPAP, monoester of PAP; DNAP, diester amide of PAP; EMS, eosinophilia myalgia syndrome. ‡
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S0893-228x(95)00181-0 CCC: $12.00
Soon thereafter, the oil associated with TOS was found to be technical-grade, aniline-denatured rapeseed oil that had been illegally sold as pure olive oil (1). Further investigations showed that aniline and fatty acid anilides were present in TOS oils, and these compounds became the first markers of toxic oils (5, 6). Other compounds were also reported to be in the disease-producing oils, although the association of these other compounds to TOS was not clear (7). Most recently, the fatty acid esters of 3-(N-phenylamino)-1,2-propanediol (PAP)sthe products of aniline and triglyceridesshave been shown to have a strong association with TOS (8). Even though the fatty acid anilides and the PAP esters have been associated with TOS, the etiological agent(s) responsible for this epidemic have not been identified. The main barrier to identification of the agent(s) has been that no animal model exists that fully duplicates TOS. In an effort to search for possible causative agents in TOS-associated oils, we used liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (LC-MS/MS) to identify more than 20 PAP esters in these oils. For the first time, we also identified a class of compounds known as the PAP ester amides in these oils as well.
Materials and Methods Samples and Standards. Oil samples, known as the Toxico-Epi-I oils, that were collected during the 1981 epidemic (2) were used in our study. Oil samples collected from the ITH refinery (the refinery most clearly associated with toxic oils) in Seville, Spain, were also used (9, 10). These include samples of two aniline-denatured rapeseed oils collected before refining and one aniline-denatured rapeseed oil collected after the refining process (8). This latter refined oil from ITH is called
© 1996 American Chemical Society
1002 Chem. Res. Toxicol., Vol. 9, No. 6, 1996
Schurz et al. Table 2. Fatty Acid Reference Table fatty acid chain
Figure 1. General structures of compounds related to the toxic oil syndrome. The structures are named after their base skeleton 3-(N-phenylamino)-1,2-propanediol (PAP). MPAP is the monoester of PAP, DPAP is the diester of PAP, and DNAP is the diester amide of PAP. The R1, R2, and R3 groups are alkyl groups associated with a specific fatty acid. Molecular weight and molecular ion information is given for each structure based upon the R1, R2, and R3 groups being C17H33 (oleyl group). Table 1. Newly Identified Components of Toxic OilssDPAPs, MPAPs, and DNAPs DPAP (Diesters) compd no.
R1(CO)
R2(CO)
(M + H)+
1 2 3a 4a 5a 6a 7a 8a 9 10 11 12 13 14
palmityl palmityl linolenyl linolenyl linolenyl linoleyl linoleyl oleyl stearyl stearyl linoleyl oleyl oleyl eicosenyl
linoleyl oleyl linolenyl linoleyl oleyl linoleyl oleyl oleyl oleyl stearyl eicosenyl eicosenyl erucyl eicosenyl
668 670 688 690 692 692 694 696 698 700 722 724 752 752
MPAP (Monoesters) compd no.
R1(CO)
(M + H)+
15 16a
linoleyl oleyl
430 432
DNAP (Diester Amides) compd no.
R1(CO)
R2(CO)
R3(CO)
(M + H)+
17 18a 19 20 21 22 23 24 25a
linoleyl linoleyl linoleyl linolenyl linoleyl oleyl oleyl linoleyl oleyl
oleyl linoleyl linolenyl oleyl linoleyl stearyl linoleyl oleyl oleyl
linolenyl linoleyl oleyl stearyl stearyl linolenyl oleyl oleyl oleyl
955 955 955 957 957 959 959 959 961
a
Denotes a standard has been synthesized for this structure.
the source-contaminated oil, and it is believed to be the primary source of all TOS oils. Before the epidemic, the refined oil was shipped to RAELCA, a packaging facility, where it was diluted with other oils and bottled for sale to the public. Using the mass spectral techniques described below, we tentatively identified probable structures for several of the compounds described in this paper. Through an agreement with the Fondo de Investigacion Sanitaria, we requested and were supplied with standards from the laboratory of Dr. Angel Messeguer of the Centro de Investigacion y Desarrollo in Barcelona, Spain (11). General structures of these compounds are shown in Figure 1, and Tables 1 and 2 provide the specific structures of all compounds. Each synthesized standard was prepared as a 10 mg/mL solution in toluene from stock solutions. Oil samples were diluted with 1-propanol to a concentration of 50 mg of oil/mL of 1-propanol. The toxicity of these substances in humans is unknown; however, they may be etiologic agents of TOS and should be treated with caution.
palmityl oleyl linoleyl linolenyl stearyl eicosenyl erucyl behenyl
C16:0 C18:1 C18:2 C18:3 C18:0 C20:1 C22:1 C22:0
molecular structure
alkyl chain
(CO)(CH2)14CH3 (CO)(CH2)7CHdCH(CH2)7CH3 (CO)(CH2)7CHdCHCH2CHdCH(CH2)4CH3 (CO)(CH2)7(CHdCHCH2)3CH3 (CO)(CH2)16CH3 (CO)(CH2)6CHdCH(CH2)10CH3 (CO)(CH2)11CHdCH(CH2)7CH3 (CO)(CH2)20CH3
C15H31 C17H33 C17H31 C17H29 C17H35 C19H37 C21H41 C21H43
Analytical Methods. Chromatography of several diesters of PAP (DPAP) was performed on a Whatman Partisil 5 ODS-3 25 cm liquid chromatography column (Whatman Inc., Fairfield, NJ) using a flow rate of 1 mL/min. Prepared oil solutions (15 µL) were injected on the column. A gradient separation was used, where solvent A was water with 0.1% acetic acid added and solvent B was methanol with 0.1% acetic acid. The initial elution conditions were 5% solvent A and 95% solvent B for 2 min, with an immediate increase to 100% solvent B, which was held for 18 min. Finally, the column was equilibrated at the starting conditions for 5 min before the next injection. Analyses and structural determinations were performed using a SCIEX API III tandem mass spectrometer system using both atmospheric pressure chemical ionization (APCI) and ionspray. Samples introduced by APCI to the desolvation chamber of the probe were nebulized by nitrogen at 80 psi and an auxiliary gas flow of 1 L/min. The desolvation chamber was heated to 500 °C, and a corona discharge was maintained at 3.0 V. Samples introduced by ionspray were infused at a rate of 10 µL/min. The ionspray needle was held at 6000 V. The instrumental conditions that were used were similar for all of the compounds. Ionspray and APCI mass spectra were identical although the collision energies and collision gas thickness sometimes differed slightly for each ionization technique. Median collision gas thicknesses were used (260-360) with argon and 10% nitrogen in the collision cell. The collision energies ranged from 86.5 to 117.0 eV, and the orifice potential was varied from 35 to 85 V. The parent ion scanning technique was used to tentatively identify new PAP esters as parents of selected daughter ions. Aniline was the denaturant in TOS-associated oils, and daughter ions containing the anilino moiety were selected to find all aniline-derived compounds in these oils. The specific daughter ions that we monitored were m/z 94 (C6H5NH3+) for the anilino moiety and m/z 132 (C6H5NHCH2CHCH+). Specific daughter ions containing fatty acid groups (examples: m/z 432 (oleyl), m/z 430 (linoleyl), and m/z 428 (linolenyl)) were used in parent ion scanning to further identify types of fatty acid esters present in the parent structure. Daughter ion mass spectrometry of selected parents was then used to derive and confirm the proposed structures. Several of these compounds were compared with synthesized standards; standards were not available for all of the identified compounds.
Results Mass Fragmentation of Esters of PAP. We used a combination of parent ion scanning techniques and daughter ion scanning techniques to find esters of PAP in TOS-associated oils. Previously, the 1,2-dioleyl DPAP, 2-(oleyloxy)-3-(N-phenylamino)propyl oleate (structure 8, Table 1), and the monoester of PAP (MPAP), 1-oleyl MPAP, 2-hydroxy-3-(N-phenylamino)propyl oleate (structure 16, Table 1), were quantitated in Toxico-Epi-I oils (2) and were found to be strongly associated with oils that caused TOS (8). On the basis of the varied fatty acid composition of the oils, we suspected that the oils contained other compounds similar in structure to compounds 8 and 16. The daughter ion spectrum of 8 and
Aniline-Triglyceride Products in TOS Oil Samples
Chem. Res. Toxicol., Vol. 9, No. 6, 1996 1003
Figure 3. Parent ion mass spectra of m/z 428, 430, and 432.
Figure 2. Ionspray MS/MS spectra of 1,2,N-trioleyl DNAP and 1,2-dioleyl DPAP.
16 had an ion at m/z 132 characteristic of those structures. Monitoring for parent ions that produced an ion at m/z 132 allowed us to identify the protonated molecular ion and characteristic retention time of each PAP ester. We then examined the daughter spectrum of each parent ion to further characterize and identify each PAP ester. Based upon these data and with comparison to authentic standards for several compounds, we found 14 diesters of PAP that are listed in Table 1 (structures 1-14), 2 monoesters of PAP (structures 15 and 16), and 8 diester amides of PAP (structures 17-25) in a refined oil from the ITH refinery, the probable source oil from which all TOS oils were derived. The fatty acid groups that are referred to in Table 1 are defined in Table 2. In addition to the compounds shown in Table 1, many of their isomers are also thought to exist in the oils, and some of these have been synthesized. The MS/MS spectra of compounds 8 and 16 were determined from standards; the spectrum of structure 8 is given in Figure 2. Several ions were common to the mass spectra of 1,2-dioleyl DPAP (structure 8, (M + H)+ ) m/z 696) and 1-oleyl MPAP (structure 16 (M + H)+ ) m/z 432). In the MS/MS spectrum of structure 8, the fragment ion at m/z 432 is formed by the elimination of the (CO)C17H33 group from the molecular ion followed by addition of a proton; this ion is similar to the molecular ion of the 1-oleyl MPAP. It can lose water to form a fragment ion at m/z 414. Loss of the side chain from the m/z 432 ion or the m/z 414 ion, followed by proton addition, forms the ions at m/z 168 (C6H5NH2CH2CHOHCH2OH+) and m/z 150. When m/z 168 loses a water molecule, it also produces the fragment ion at m/z 150. Similarly, loss of a water molecule from the m/z 150
produces the very strong fragment ion at m/z 132 (C6H5NHCHCHCH2+); this ion was used for quantitation of the DPAP. Related to the previously described losses is the ion at m/z 106, representing (C6H5NHCH2+). The fatty acid chain, C17H33 (CO)+, is observed at m/z 265 in the daughter ion spectrum and, if sufficient collision energies are used, will break down into smaller fragment ions separated by 13 and 14 amu. Parent Ion Mass SpectrometrysDPAPs. To identify new DPAPs and MPAPs that are structurally similar to structures 8 and 16, we analyzed the case oil containing the greatest amount of these compounds using parent ion mass spectrometry as described in the Materials and Methods. Parent ion mass spectrometry sets the analyzing quadrupole (Q3) of a triple stage quadrupole mass spectrometer to a single mass (a known daughter), selects ions one mass at a time with the first quadrupole (Q1), and sends those ions into the collision cell (Q2). In Q2, the selected ions collide with argon which causes fragmentation to daughter ions; Q3 mass analyzes the fragments produced in Q2. If the measured daughter ion mass agrees with the fixed Q3 mass (predetermined), the operating system will record the mass of the parent that was first transmitted to the collision cell from Q1. For the identification of compounds structurally similar to DPAP, the daughter ion m/z 132 was selected. This daughter ion does not contain any fatty acid ester groups and would identify the largest number of DPAP compounds independent of the type of fatty acid ester group present. The initial parent ion scan, using m/z 132 as the fragment ion, identified several parent ions (m/z 688, 690, 692, 694, 696, 722, 724, 752) that could be tentatively identified as diester DPAP structures. Because it is possible to have more than one species being selected (i.e., isomers) in a parent ion scan, further identification of the molecular ion must be done using a combination of techniques. The first of these uses parent ion mass spectrometry to obtain more specific information on the identities of these compounds on the basis of the fragmentation of a standard. The second uses daughter ion mass spectrometry of the molecular ion. As described earlier, the daughter ion mass spectrum of 1,2-dioleyl DPAP contained a fragment ion at m/z 432 that was produced by the loss of the (CO)C17H33 group. This type of loss is reflected in the daughter ion mass spectra of all DPAPs. Figure 3 contains the parent ion scans of three daughter ions formed by a similar loss: m/z 428 (linolenyl containing fragment), 430 (linoleyl containing fragment), and 432 (oleyl containing fragment). The parent ion scan of m/z 432 revealed three possible parent ions with masses m/z 696, 694, and 692.
1004 Chem. Res. Toxicol., Vol. 9, No. 6, 1996
Figure 4. MS/MS spectra of 1-linoleyl-2-oleyl DPAP and 1-oleyl-2-linoleyl DPAP.
The parent ion scan of m/z 430 produced parent ions at m/z 694, 692, and 690. The weakest spectrum indicated only two parent ions for the m/z 428 daughter ion at m/z 692 and 690. Knowing the parent ion and the daughter ion masses, it is possible to determine the missing part of the molecule. For example, the parent ion scan of m/z 432 (an oleyl containing fragment of DPAP) has a parent ion mass of m/z 696. This is a difference of 264 amu, which corresponds to a loss of the C17H33(CO) group and addition of a proton. This originates from the oleyl group, indicating that the parent ion m/z 696 is probably composed of two oleyl groups. The daughter ion mass spectra of m/z 696 in the actual oil sample and the standard were identical, indicating that m/z 696 is likely to be composed of only the dioleyl DPAP. Parent ion MS/ MS spectra were measured for fragments containing other fatty acid groups (stearyl, m/z 434; palmityl, m/z 406; erucyl, m/z 488; behenyl, m/z 490; and eicosenyl, m/z 460) in order to identify other diesters of PAP. The DPAPs (structures 1-14) identified using the abovedescribed experiments are compiled in Table 1. Structural Isomers of PAP. Compounds 3, 6, 8, 10, and 14 are composed of two identical fatty acids (R1(CO) ) R2(CO)). Among the diester structures were several molecules that contained different fatty acid groups. To determine the position of the fatty acid groups in the structural isomers of the DPAPs, we studied the mass spectra of several structural isomers (Table 1, compounds 4, 5, and 7). The structural isomers of compounds 4, 5, and 7, when present in solution together, could not be separated by reverse-phase chromatography; however, they could be distiguished based on their mass spectra. Figure 4 contains the mass spectra of two synthesized diester standards, 1-linoleyl-2-oleyl DPAP, 7, and 1-oleyl-2linoleyl DPAP. The daughter ions formed by the loss of the R2(CO) group are consistently of greater intensity than the the loss of the R1(CO) group, indicating a preferential loss of the R2(CO) group. For example, in the MS/MS spectrum of 1-linoleyl-2-oleyl DPAP the daughter ion m/z 430 (formed by the loss of the oleyl group at the C2 position) has greater response than the daughter ion at m/z 432. The converse of this observation is true in the MS/MS spectrum of 1-oleyl-2-linoleyl DPAP. Since we could not separate the combined isomers chromatographically in the oils or as standards using our LC method, we examined the DPAP standards and the ITH refined oil by flow injection. The parent ion mass
Schurz et al.
(m/z 694 for 1-oleyl-2-linoleyl DPAP and 1-linoleyl-2-oleyl DPAP, for example) and the primary daughter ion of each isomer (m/z 432 and 430) were monitored. Solutions of equal and unequal concentrations of both isomer forms were prepared and evaluated. These solutions indicated that it would be possible to determine whether a particular isomer predominated on the basis of the response of its major fragment. When the ITH-refined oil was evaluated, however, the measurement of the parent ion to daughter ion had an equivalent response for both daughter ions. This response was also obtained in standards when the isomer forms had equivalent concentrations. We also found these results using solutions of standards of compounds 4 and 5 for which we had both isomer forms synthesized. This suggests that the isomer forms are probably present at equal concentration in the oils, and although we listed 14 different DPAPs, it seems likely that there may be 23 different DPAPs where R1 and R2 are reversed for compounds 1, 2, 4-7, 9, and 1113. Further research is required to elucidate these isomeric compounds. Estimated Concentrations of Diesters of PAP in ITH Refined Oil. The major DPAP (non-isomer) species were easily separated by a C18 liquid chromatography column. A suitable internal standard was not available at the time these were examined, so the concentrations of the DPAPs were estimated relative to the concentration of the 1,2-dioleyl DPAP (structure 8) in the sourcecontaminated oil (i.e., ITH-refined oil). The major DPAPs that corresponded to m/z 694 (1-linoleyl-2-oleyl DPAP, structure 7), m/z 692 (1-linolenyl-2-oleyl DPAP, structure 5, and 1,2-di-linoleyl DPAP, structure 6), m/z 690 (1linolenyl-2-linoleyl DPAP, structure 4), and m/z 688 (1,2di-linolenyl DPAP, structure 3) were measured simultaneously with m/z 696 (1,2-dioleyl DPAP, structure 8) in the source-contaminated oil. Individual isomers and different species were measured together without chromatographic separation. The concentration values were reported as total estimated concentrations of the m/z values of the DPAPs. Since the 1,2-dioleyl DPAP (structure 8) concentration had already been measured in the ITH-refined oil, it was used as an internal standard in these measurements; the other DPAP structures were assumed to have a similar response as the 1,2-dioleyl DPAP. In the source-contaminated oil, the concentration of 1,2-dioleyl DPAP was 150 ppm. The following concentrations were measured relative to the concentration of 1,2-dioleyl DPAP: m/z 694 ) 300 ppm, m/z 692 ) 150 ppm, m/z 690 ) 37.5 ppm, and m/z 688 ) 18.8 ppm. These concentrations indicate that the total DPAP concentration is approximately 650 ppm in the ITH-refined oil. Other DPAP structures, not mentioned above but listed in the table, had a much smaller response than those described above; therefore, their concentrations have not been determined. We are waiting for standards to make that determination. Parent Ion Mass SpectrometrysMonoesters of PAP. The monooleyl MPAP (structure 16, Table 1) was also detected in the refined oil from ITH. The daughter ion spectrum of 1-oleyl MPAP ((M + H)+ ) m/z 432) has similarities to the 1,2-dioleyl DPAP MS/MS spectrum. Significant daughter ions present here include the ion that is produced by the loss of the aniline group (C6H5NH2) from the molecular ion forming m/z 339. The aniline-containing group is present in the fragment ion at m/z 106. Another significant fragment ion is produced by the loss of an oleyl group (C17H33(CO)) with the
Aniline-Triglyceride Products in TOS Oil Samples
concurrent loss of 2 water molecules to form the fragment ion at m/z 132. The oleyl group (C17H33(CO)+) is detected at m/z 265. As is the case with the daughter ion mass spectrum of the DPAP, fragmentation of the oleyl chain occurs when collision energies are increased producing fragment ions 13 and 14 amu apart. A second MPAP (structure 15, Table 1) that corresponds to the 1-linoleyl MPAP ((M + H)+ ) 430) was detected using parent ion scans of m/z 132. We have assumed that the position of the fatty acid group on these molecules is R1(CO), although a combination of both may exist in the oils as structural isomers. Parent Ion Mass SpectrometrysDiester Amides of PAP. While we were determining the structures of the DPAPs and MPAPs, we discovered another group of related compounds. These structures are referred to as diester amides of PAP (DNAP, Figure 1), owing to formation of an amide at the nitrogen of the DPAP. Tentative identifications of several structures were made; and the 1,2,N-trioleyl DNAP ((M + H)+ 961), 1,2,Ntrilinoleyl DNAP ((M + H)+ 955), 1,2,N-trilinolenyl DNAP ((M + H)+ 949), and 1,2,N-tristearyl DNAP ((M + H)+ 967) were synthesized. Of those standards only the 1,2,N-trioleyl DNAP (structure 25) and 1,2,N-trilinoleyl DNAP (structure 18) were confirmed in toxic oils. Similar to the DPAPs, the parent ions and daughter fragment ions suggest that combinations of different fatty acid groups make up the variety of DNAPs that are observed (Table 1, structures 17-25), and it seems likely that there are structural isomers present. Though exact mass measurements of these compounds were not performed, their exact masses were calculated. This was done because the nominal mass of the 1,2,N-trioleyl DNAP, MW ) 959 amu, is in disagreement with the molecular ion (M + H)+ ) m/z 961. This apparent mass discrepancy is due to 13C isotope and the relatively large size of the molecule. The exact mass of 1,2,N-trioleyl DNAP is 959.83 amu, almost 1 amu greater than its nominal mass. Therefore, in a unit mass resolving instrument (i.e., a quadrupole mass spectrometer) the (M + H)+ ) m/z 961. This is true for all of the DNAP species. The DNAPs, indicated by parent ions at m/z 961 and m/z 959, were initially detected in parent ion scans of the molecular ion for 1,2-dioleyl DPAP, 8 (m/z 696). The daughter ion mass spectra of these two ions produced not only a DPAP-like fragment but similar fragmentation patterns as well. Further parent ion scans were performed that indicated the presence of several other possible DNAP structures, in particular, m/z 955, 957, and 959, in addition to m/z 961. Chromatography confirmed that the DPAPs and the DNAPs were discrete compounds and that DPAP was not simply a daughter of DNAP. The procedure for identifying the various fatty acid groups was similar to that used for identifying the DPAPs. The most abundant daughter ion in the MS/MS spectrum of 1,2,N-trioleyl DNAP, 25, is the m/z 678 ion (Figure 2). This ion is formed by the loss of the acid R2CO2H and structurally is the same as the m/z 678 produced in the spectrum of 1,2-dioleyl DPAP. The daughter ion at m/z 696 is first formed by the loss of the R2 (CO) from the (M + H)+ parent ion at m/z 961. Formation of the m/z 678 ion is achieved by the loss of a water molecule by reduction of the hydroxyl group. The remaining daughter ions have the same structural identity as the DPAP structures.
Chem. Res. Toxicol., Vol. 9, No. 6, 1996 1005
Using MS/MS analysis to characterize a suspected DNAP, we determined the identity of the ester group at the C2 carbon, which is simply the difference between the parent ion mass (i.e., m/z 961) and the mass of the DPAP-like fragment ion at m/z 696, i.e., 961 - 696 ) 265 (C17H33(CO)). A more abundant ion was most often used in this determination, however, instead of the DPAP-like fragment ion. That ion was m/z 678, formed by the loss of the fatty acid chain and a water molecule. The loss of two fatty acid groups (2 C17H33(CO)) and the addition of a proton formed a MPAP-like fragment ion (m/z 432); this loss helped identify the ester group at the C1 carbon and verified the group at the C2 carbon. The alkyl group located at the nitrogen is determined by subtraction once the groups at C1 and C2 are known. As in the DPAP structures, it is likely that structural isomers exist for these compounds, especially at the C1 and C2 carbon atoms. DNAP structures suspected in the oils are listed in Table 1 (structures 17-25). DNAPs 18 and 25 were confirmed, and mass spectral identification suggested that DNAPs 17-23 were also present. We did not have standards of these DNAPs for confirmation of their structures. Simpler fatty acid anilides are also present in the oils, as well as the more complex structures described in this paper. In earlier work, Bernert et al. (3) measured the oleyl, linoleyl, and palmityl anilide content of a set of carefully selected oils. In conjunction with the work presented here, the concentrations of linolenyl, stearyl, eicosenyl, erucyl, and behenyl anilides were measured in the same set of oils. Of those compounds, the eicosenyl, erucyl, and behenyl anilides were not previously identified as components of the contaminated oils. These compounds were found to be associated with TOSassociated oils, but not as highly associated with case oils that caused the disease as the previously mentioned DPAPs and MPAPs.
Discussion The presence of DPAPs, MPAPs, and DNAPs in TOSassociated oils is an important finding. These compounds are very similar in structure to principal components of cell membranes, the diacylglycerols. If these compounds were incorporated into cell membranes, their presence could evoke an immunologic response. These compounds seem to be good candidates for etiologic agents of TOS (8). Until we have a clear understanding of the cause of TOS, we cannot prevent outbreaks of similar disease in the future. This is evidenced by the 1989 epidemic in the United States that became known as the eosiniphilia myalgia syndrome (EMS; 12). That disease was associated with the consumption of L-tryptophan food supplements, and like TOS, its etiologic agent(s) have not been identified. For TOS, we have now identified several new compounds that are markers for oils that cause disease, and that are candidates for animal and toxicological testing to determine whether some of the compounds may be etiologic agents. These compounds are products of aniline and triglycerides and come in the form of fatty acid anilides, diesters of DPAP (14 structures), monoesters of MPAP (2 structures), and diester amides or DNAPs (9 structures). What draws both TOS and EMS together is that clinically, they are strikingly similar (12-15). This clinical similarity suggests that a common biological pathway was affected by both the TOS-associated case
1006 Chem. Res. Toxicol., Vol. 9, No. 6, 1996
oils and the L-tryptophan case samples, and that this commonality may provide an answer to what caused the diseases. Recently, researchers from the Mayo Clinic have proposed a possible link between TOS and EMS (16). They reported the in vitro conversion of (Nphenylamino)alanine, an EMS-associated contaminant, to 3-(N-phenylamino)-1,2-propanediol. The latter compound forms the basic skeleton of the DPAPs, MPAPs, and DNAPs. Apart from the clinical similarities between TOS and the EMS, both diseases seem to damage the immune system and to elicit a response. People with TOS and EMS still suffer from their disease in a chronic phase that includes peripheral neuropathy, hepatopathy, scleroderma, and pulmonary hypertension. Many times their disease will include myalgias and arthralgias. In this chronic phase, the disease takes on the appearance of a rheumatic illness, particularly of scleroderma (17). It has been suggested that scleroderma and related disorders may be triggered by exposure to some environmental agent(s) when a pre-existing susceptibility is present in the victim (18). Certain host factors (e.g., age, immunogenetic background) may have played a role in determining the susceptibility of the victim to the illness. The study of TOS and EMS may shed light on the etiology and pathogenesis of not only these diseases but of a whole host of other illnesses. Perhaps the greatest challenge in identifying the causes of TOS and EMS lies in the fact that no suitable animal models have been identified that simulate TOS or EMS. All compounds that are identified as possible etiologic agents must be evaluated as toxic agents. With the lack of an animal model it will be very difficult, if not impossible, to determine whether a compound is the actual etiologic agent. It is imperative that research on these diseases continues until suitable animal models, and perhaps a link between TOS and EMS, have been identified and the etiologic agents of both diseases are known.
Acknowledgment. We thank Dr. Angel Messeguer for providing synthesized standards of the various diesters and diester amides. The authors would also like to thank the continued support of the Government of Spain, which has allowed us to continue this research. This work was supported in part through an international collaborative agreement with the Fondo de Investigacion Sanitaria of the Government of Spain. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or the U.S. Department of Health and Human Services.
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