A New Conjugated Linoleic Acid Isomer, 7 trans, 9 cis- Octadecadienoic Acid, in Cow Milk, Cheese, Beef and Human Milk and Adipose Tissue

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1 A New Conjugated Linoleic Acid Isomer, 7 trans, 9 cis- Octadecadienoic Acid, in Cow Milk, Cheese, Beef and Human Milk and Adipose Tissue Martin P. Yurawecz a, *, John A.G. Roach a, Najibullah Sehat a, Magdi M. Mossoba a, John K.G. Kramer b,1, Jan Fritsche c, Hans Steinhart c, and Youh Ku a a U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Washington, DC 20204, b Southern Crop Protection, Food Research Center, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada, N1G2W1, and c Institute of Food Chemistry, University of Hamburg, Hamburg, Germany ABSTRACT: The identity of a previously unrecognized conjugated linoleic acid (CLA) isomer, 7 trans, 9 cis-octadecadienoic acid (18:2) was confirmed in milk, cheese, beef, human milk, and human adipose tissue. The 7 trans, 9 cis-18:2 isomer was resolved chromatographically as the methyl ester by silver ion high-performance liquid chromatography (Ag + HPLC); it eluted after the major 9 cis, 11 trans-18:2 isomer (rumenic acid) in the natural products analyzed. In the biological matrices investigated by Ag + HPLC, the 7 trans, 9 cis-18:2 peak was generally due to the most abundant minor CLA isomer, ranging in concentration from 3 to 16% of total CLA. By gas chromatography (GC) with long polar capillary columns, the methyl ester of 7 trans, 9 cis-18:2 was shown to elute near the leading edge of the major 9 cis, 11 trans-18:2 peak, while the 4,4-dimethyloxazoline (DMOX) derivative permitted partial resolution of these two CLA isomers. The DMOX derivative of this new CLA isomer was analyzed by gas chromatography electron ionization mass spectrometry (GC EIMS). The double bond positions were at 7 and 9 as indicated by the characteristic mass spectral fragment ions at m/z 168, 180, 194, and 206, and their allylic cleavages at m/z 154 and 234. The cis/trans double-bond configuration was established by GC direct deposition Fourier transform infrared as evidenced from the doublet at 988 and 949 cm 1 and absorptions at 3020 and 3002 cm 1. The 7 trans, 9 cis-18:2 configuration was established by GC EIMS for the DMOX derivative of the natural products examined, and by comparison to a similar product obtained from treatment of a mixture of methyl 8-hydroxy- and 11-hydroxyoctadec-9 cis enoates with BF 3 in methanol. Lipids 33, (1998). *To whom correspondence should be addressed at Office of Food Labeling, HFS-175, U.S. Food and Drug Administration, 200 C St., S.W., Washington DC mpy@cfsan.fda.gov 1 Contribution number S010 from the Food Research Center, Guelph, Ontario, Canada. Abbreviations: Ag + HPLC, silver ion high-performance liquid chromatography; cis/trans, refers to all the CLA isomers having either a cis,trans or a trans,cis configuration; CLA, conjugated linoleic acid; DMOX, 4,4-dimethyloxazoline; FAME, fatty acid methyl esters; GC DD FTIR, gas chromatography direct deposition Fourier transform infrared; GC EIMS, gas chromatography electron ionization mass spectrometry. The major conjugated linoleic acid (CLA) isomer present in cow milk (1 9), human milk (10,11), cheese (2,3,5,12 16), beef (17), and human adipose tissue (18,19) was reported to be 9 cis, 11 trans-octadecadienoic acid (18:2), or rumenic acid. CLA is a collective term referring to all conjugated geometric and positional isomers of 18:2. Parodi (1) determined the major 9 cis, 11 trans-18:2 isomer in milk by a combination of gas chromatography (GC) and argentation thin-layer chromatography, partial hydrazine reduction, and ozonolysis. In addition to the 9 cis, 11 trans-18:2, Ha et al. (2) reported seven more CLA isomers (10 cis, 12 trans-18:2; 10 trans, 12 cis-18:2; 11 cis, 13 cis-18:2; 9 cis, 11 cis-18:2; 10 cis, 12 cis-18:2; and 9 trans, 11 trans-18:2/10 trans, 12 trans-18:2) in cheese based on comparing equivalent chain length data with published results (20), and GC chemical ionization mass spectrometry of the fatty acid methyl esters (FAME). Recently, Lavillonnière et al. (16) identified five additional minor CLA isomers in cheese (8 cis, 10 trans-18:2; 8 cis, 10 cis-18:2; 8 trans, 10 trans-18:2; 11 trans, 13 trans-18:2; and 11?,13?-18:2). They partially reduced the CLA mixture with hydrazine, isolated the resulting monounsaturated fatty acids by silver nitrate thin-layer chromatography and identified the fatty acids by gas chromatography electron ionization mass spectrometry (GC EIMS) as their 4,4-dimethyloxazoline (DMOX) derivatives. These authors also evaluated the total CLA mixture by GC EIMS. In the past decade, unequivocal identification of CLA isomers was very limited for the following reasons. Firstly, in almost all reports, the identifications of GC peaks were solely based on comparing observed GC retention times with those of ill-defined commercial CLA mixtures, without independent confirmation of provisional assignments by chemical or spectroscopic methods. Secondly, acid-catalyzed methylation procedures, which were reported to increase trans,trans CLA isomers (8), were not avoided. Thirdly, even with 100-m GC capillary columns, the resolution of all the CLA isomers has not been achieved (16,21). Therefore, other confirmatory techniques were necessary to identify CLA isomers. Diagnostic ions in the EI mass spectra observed for a mixture of the 4-phenyl-1,2,4-triazoline-3,5-dione adducts (22) were suc- Copyright 1998 by AOCS Press 803

2 804 M.P. YURAWECZ ET AL. cessfully used to identify double-bond positions in CLA mixtures. GC EIMS with selected ion monitoring was used to suggest the presence of different CLA isomers in an unresolved mixture of four 4-methyl-1,2,4-triazoline-3,5-dione adducts of CLA (23). Full mass scan data allowed the identification of individual isomers in conjugated fatty acid mixtures resolved by GC of 4-methyl-1,2,4-triazoline-3,5-dione derivatives (24). Reconstructed ion profiles of the isomer-specific ions for CLA DMOX were used to distinguish between the signals of different CLA isomers in the present study. This permitted the determination of double-bond positions for partially resolved CLA isomers. Recently, we modified a silver ion high-performance liquid chromatography (Ag + HPLC) method by Adlof et al. (25). For the first time, using the modified method, we separated 12 geometric and positional CLA isomers found in a commercial mixture (15). This proved to be an essential complementary chromatographic technique for the identification of CLA isomers. In the present communication, a newly identified CLA isomer found in milk, cheese, beef, human milk, and adipose tissue matrices was resolved as the FAME derivative by Ag + HPLC, and as the DMOX derivative by GC. This CLA isomer was identified as 7 trans, 9 cis-18:2 by GC EIMS and GC-direct deposition-fourier transform infrared (GC DD FTIR) spectroscopy. The trans,cis geometry was established by GC EIMS for the DMOX derivative of the natural products examined, and by comparison with a known 7 trans, 9 cis-18:2 product. Preparation of FAME. The total lipids from milk, cheese, beef, and human adipose tissue (20 70 mg) were methylated using anhydrous NaOCH 3 /methanol (15). The FAME were analyzed directly by GC and Ag + HPLC. The free fatty acids were methylated with a 10% trimethylsilyldiazomethane as described previously (15,27). Ag + HPLC. The HPLC equipment and conditions were the same as described previously (15). A ChromSpher 5 Lipids semipreparative (10 mm i.d. 250 mm stainless steel; 5 µm particle size) and an analytical (4.6 mm i.d. 250 mm stainless steel; 5 µm particle size) silver impregnated columns were used (Chrompack, Bridgewater, NJ). The mobile phase was 0.1% acetonitrile in hexane and was operated isocrati- MATERIALS AND METHODS A mixture of CLA isomers was purchased from Nu-Chek- Prep, Inc. (Elysian, MN). Several pure CLA isomers were obtained as their free fatty acids from Matreya Inc. (Pleasant Gap, PA). Acetonitrile and hexane were ultraviolet grade. Other solvents were distilled-in-glass quality. 2-Amino-2- methyl-1-propanol (95%) was purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI). A 10% solution of trimethylsilyldiazomethane (TMS-DAM) in hexane was obtained from TCI America (Portland, OR). Anhydrous NaOCH 3 /methanol was purchased from Supelco, Inc. (Bellefonte, PA). Cow milk was available from a previous study (9). Cheese and beef products were purchased locally. The biological specimens were human subcutaneous adipose tissues. They were a gift from Dr. H.J. Boehles (Johann-Wolfgang- Goethe University Clinic, Frankfurt, Germany) and were obtained from a group of male and female children, aged 1 13 yr, with inguinal hernia. Human milk was a gift from Dr. G. Jahreis (Friedrich Schiller University, Jena, Germany). Lipid extraction. Milk lipids were extracted using a modified Folch procedure as described previously (8). Cheese was extracted with diethyl ether/petroleum ether (1:1) after homogenization with ethyl alcohol in the presence of potassium oxalate. The total cheese lipids were then dried over anhydrous Na 2 SO 4. Beef lipids were extracted with chloroform/methanol as described previously (26). Human adipose tissue was extracted as described previously (19). FIG. 1. Partial gas chromatography electron ionization mass spectrometry (GC EIMS) chromatogram of conjugated fatty acid 4,4-dimethyloxazoline (DMOX) derivatives from human adipose tissue showing the reconstructed ion profiles for m/z 333 (top); and m/z 234 and 262 (bottom). In the cis/trans region, the first two peaks (m/z 234) were due to 7 cis, 9 trans-18:2, 7 trans, 9 cis-18:2, respectively, and the third one (m/z 262) was due to 9 cis, 11 trans-18:2.

3 7 TRANS, 9 CIS-18:2 IN BIOLOGICAL MATRICES 805 FIG. 2. GC EIMS spectrum of the DMOX derivative of 7 trans, 9 cis-18:2 from cow milk. For abbreviations see Figure 1. cally. The flow rates were 4.0 ml/min and 1.0 ml/min, for the semipreparative and analytical columns, respectively. Detection was at 233 nm. DMOX derivatives. DMOX derivatives were prepared as described previously (15,28,29). Instrumentation. The GC (8), GC EIMS (15,28), and GC DD FTIR (30) instruments and conditions were given previously. RESULTS AND DISCUSSION The m/z 333 GC EIMS ion profile for the DMOX derivatives of fatty acids from human adipose tissue (Fig. 1 top) showed the presence of a small peak emerging just ahead of that of the major 9 cis, 11 trans-18:2 isomer. A similar m/z 333 GC- EIMS ion profile was observed for the fatty acid DMOX derivatives of all other natural matrices investigated. The mass spectrum for this new peak is shown in Figure 2 for cow milk. The EI mass spectrum of the new compound exhibited an abundant molecular ion at m/z 333, characteristic of 18:2 DMOX derivatives. An abundant ion at m/z 126, followed by a homologous series of ions separated by gaps of 14 mass units, with intervals of 12 mass units found between m/z 168 (C6) and 180 (C7), and between m/z 194 (C8) and 206 (C9), indicated the presence of two double bonds at 7 and 9. In addition, the mass spectrum showed two favorable allylic cleavage ions at m/z 154 and 234, supporting a 7,9-18:2 structure. The abundant ion at m/z 152 is due to an anomaly (31), which remains unexplained. The reconstructed ion profile for the characteristic allylic ion m/z 234 showed two peaks due to 7,9-18:2 isomers that eluted before the one at m/z 262 for the 9 cis, 11 trans-18:2 isomer in Fig. 1 (bottom). The possible geometric configuration of the two 7,9-18:2 isomers will be discussed below. These results demonstrated that the first two eluting peaks (m/z 234) did not arise from the adjacent major 9 cis, 11 trans-18:2 isomer (m/z 262). Techniques, such as GC equivalent chain length (2), and partial hydrazine reduction followed by GC separation of the resultant 18:1 iso-

4 806 M.P. YURAWECZ ET AL. FIG. 3. Gas chromatography direct deposition Fourier transform infrared (GC DD FTIR) spectrum of 7 trans, 9 cis-18:2 DMOX derivative from cheese. For other abbreviation see Figure 1. meric mixtures (16), did not identify the 7,9-18:2 isomer in cheese. A possible reason for the latter is that the 7 trans-18:1 FAME coeluted with the 6 and 8 trans-18:1 FAME, and the 7 trans-18:1 DMOX coeluted with the 9 trans-18:1 DMOX, even on long polar capillary columns (32). The geometric configuration of the 7,9-18:2 DMOX derivatives was established by GC DD FTIR, as shown for cheese in Figure 3. The observed IR spectrum exhibited the characteristic doublet for conjugated cis/trans dienes at 988 and 949 cm 1 and absorptions at 3020 and 3002 cm 1 (19,33). The IR spectrum of 7,9-18:2 was similar to that of 9 cis, 11 trans- 18:2. However, FTIR was not able to distinguish between a cis,trans or a trans,cis configuration. Ag + HPLC was shown to separate the different trans,trans, cis/trans, and cis,cis CLA isomers of 8,10-18:2, 9,11-18:2, 10,12-18:2, and 11,13-18:2 (15), and is shown for comparison in Figure 4 (top). The 7,9-18:2 isomer in the natural matrices investigated was clearly separated by Ag + HPLC, eluting in the cis/trans region after the major 9 cis, 11 trans-18:2 peak, and was generally the most abundant of the minor CLA positional isomers. Figure 4 shows typical Ag + HPLC separations for cow milk, cheese, beef, and human adipose tissue and human milk. The relative elution position of this peak is consistent with a cis/trans configuration found by IR for this compound. To demonstrate that this peak was not due only to the 8 trans, 10 cis-18:2 isomer (Fig. 4, top), which eluted at about the same retention volume as that of the cis/trans 7,9-18:2 peak (Fig. 4, bottom five chromatograms), a representative cheese sample was fractionated as FAME by semipreparative Ag + HPLC. The fraction enriched in the cis/trans 7,9-18:2 isomer was further analyzed as the DMOX derivative by GC EIMS. The results obtained were similar to those shown in Figures 1 and 2, thus confirming the double bond positions of this new isomer. The difference in retention time between the 8 trans, 10 cis-18:2 and trans/cis 7,9-18:2 was also demonstrated chromatographically by coinjecting increasing amounts of a commercial CLA mixture (Fig. 5A) added to a beef fat extract (Fig. 5B). With increased amounts of the commercial CLA mixture, the 8 trans, 10 cis-18:2 emerged as the more abundant and before the 7,9-18:2 CLA isomer in the coinjected mixture (Fig. 5, bottom). These data do not indicate the presence or absence of the 8 trans, 10 cis-18:2 isomer in the natural products investigated. The absolute configuration of the 7,9-18:2 isomer was established by using two methods. The DMOX derivatives of geometric CLA isomers were separated by GC (19); the 9 cis, 11 trans-18:2 eluted before the 9 trans, 11 cis isomer (19). Based on this evidence, the first of the two m/z 234 peaks in Fig. 1 (bottom) was due to 7 cis, 9 trans-18:2, while the sec-

5 7 TRANS, 9 CIS-18:2 IN BIOLOGICAL MATRICES 807 FIG. 4. Silver ion high-performance liquid chromatography (Ag + HPLC) chromatograms of a commercial conjugated linoleic acid (CLA) mixture containing 12 CLA isomers and of cow milk, cheese, beef, human adipose tissue, and human milk. Concentration-dependent variations in resolution were observed between the 9,11-18:2 and the 7,9-18:2 cis/trans isomers, but the elution sequence was not altered. ond, more abundant, peak was due to 7 trans, 9 cis-18:2. Further independent confirmation was obtained from a mixture of methyl 8-hydroxy- and 11-hydroxyoctadec-9-cis-enoates treated with BF 3, as reported previously (28). The products of this reaction included two 7,9-18:2 isomers: 7 trans, 9 cis- 18:2 and 7 trans, 9 trans-18:2 (28). Analysis of this mixture as DMOX derivatives by GC showed that the 7 trans, 9 cis- 18:2 coeluted with the peak of the new CLA isomer (data not shown), thus establishing its identity. A similar result was also found for the 7 trans, 9 trans-18:2 (vide infra). Ag + HPLC did not separate the cis/trans geometric isomers. In addition, Figure 1 is also consistent with the presence of trace levels of 7 trans, 9 trans-18:2, 7 cis, 9 trans-18:2, and 7 cis, 9 cis-18:2, based on the GC EIMS reconstructed ion profile of the allylic ion m/z 234 for the DMOX derivatives of human adipose tissue. This recontructed ion profile permitted the identification of the 7 trans, 9 trans-18:2 isomer that was FIG. 5. Ag + HPLC chromatographic distinction between the 7 trans, 9 cis-18:2 isomer found in total beef fat and the 8 trans, 10 cis-18:2 isomer present in a commercial CLA mixture. Coinjection of these mixtures indicated that 7 trans, 9 cis-18:2 elutes after 8 trans, 10 cis-18:2. For abbreviations see Figure 4. not resolved by GC. Furthermore, the last eluting trans, trans Ag + HPLC peak observed for the different specimens investigated had the same retention volume as that of the 7 trans, 9 trans-18:2 product of the BF 3 reaction with methyl 8-hydroxyoctadec-9-cis-enoate. This result was consistent with the presence of a trace of 7 trans, 9 trans-18:2 in these matrices. The identities of minor CLA isomers will need to be fully confirmed by direct measurements. The relative abundance of the 7 trans, 9 cis-18:2 isomer in a few select examples of natural products was determined by Ag + HPLC (Table 1). In conclusion, a new CLA isomer was found in milk (cow and human), cheese, beef, and human adipose tissue, and it was generally prominent among the minor CLA isomers ranging in concentration from 3 to 16% of the total CLA isomers. This CLA isomer would therefore also be expected in all ruminants and dairy products, and in humans and animals that consume them. The isomer was shown to be 7 trans, 9 cis-18:2 based on GC EIMS, GC DD FTIR and Ag + HPLC results. These results emphasize the need to use the most polar GC columns available, such as long CP-Sil 88 or

6 808 M.P. YURAWECZ ET AL. TABLE 1 Amount of 7,9-18:2 FAME Isomer, Relative to Total CLA FAME, in Natural Products as Determined by Ag + HPLC a CLA Isomers (% of total CLA) Natural product 9c,11t 7t,9c Other c/t Total t/t Human milk b Cow milk b Sheep milk Cheese c Beef Human adipose tissue b a Abbreviations: FAME, fatty acid methyl ester; CLA, conjugated linoleic acid; Ag + HPLC, silver ion high-performance liquid chromatography. b Ranges obtained for five different samples. c Ranges obtained for five types of cheese. SP2560 capillary columns, to resolve as many CLA isomers as possible (21). Furthermore, we found it essential to confirm the identity of CLA isomers by GC EIMS as their DMOX derivatives. Optimal GC conditions (21) allowed the resolution of 7 trans, 9 cis-18:2 and 9 cis, 11 trans-18:2 as DMOX derivatives. Erroneous conclusions may result if CLA assignments are solely based on chromatographic retention times. Ag + HPLC will become a necessary complementary method for the identification of the different geometric and positional CLA isomers including those present at trace levels (15). The 7 trans, 9 cis-18:2 isomer in biological matrices and dairy products may arise by desaturation of the 7 trans- 18:1 as demonstrated by Pollard et al. (34). REFERENCES 1. Parodi, P.W. (1997) Conjugated Octadecadienoic Acids of Milk Fat, J. Dairy Sci. 60, Ha, Y.L., Grimm, N.K., and Pariza, M.W. (1989) Newly Recognized Anticarcinogenic Fatty Acids: Identification and Quantification in Natural and Processed Cheeses, J. Agric. Food Chem. 37, Chin, S.F., Liu, W., Storkson, J.M., Ha, Y.L., and Pariza, M.W. (1992) Dietary Sources of Conjugated Dienoic Isomers of Linoleic Acid, a Newly Recognized Class of Anticarcinogens, J. Food Comp. Anal. 5, Henninger, M., and Ulberth, F. 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