Magdi Mossoba - Academia.edu (original) (raw)
Papers by Magdi Mossoba
Journal of The American Oil Chemists Society, 1993
Interest in conjugated-diene fatty acids in foods has recently been increased by discovery of the... more Interest in conjugated-diene fatty acids in foods has recently been increased by discovery of their antioxidant and anticarcinogenic properties. Conjugated octadecatrienes (COTs), members of another group of fatty acids, are also present in foods. COTs are formed during the processing of vegetable oils as the result of the dehydration of secondary oxidation products of linoleic acid. Little information is available
Journal of High Resolution Chromatography, 1999
A commercial mixture of conjugated linoleic acid (CLA) isomers, reportedly consisting of six comp... more A commercial mixture of conjugated linoleic acid (CLA) isomers, reportedly consisting of six components, was recently resolved into 12 peaks attributed to CLA isomers using silver-ion high performance liquid chromatography (Ag + -HPLC). In this study, the coupling of two ...
Journal of High Resolution Chromatography, 1999
A commercial mixture of conjugated linoleic acid (CLA) isomers, reportedly consisting of six comp... more A commercial mixture of conjugated linoleic acid (CLA) isomers, reportedly consisting of six components, was recently resolved into 12 peaks attributed to CLA isomers using silver-ion high performance liquid chromatography (Ag + -HPLC). In this study, the coupling of two ...
Vibrational Spectroscopy, 2005
Fourier transform infrared (FTIR) spectroscopy has been established as a rapid bacteria identific... more Fourier transform infrared (FTIR) spectroscopy has been established as a rapid bacteria identification technique. To increase the throughput of this methodology, the feasibility of applying contact deposition microarray technology to print intact bacterial cells as arrayed deposits (150 mm diameter) on optical substrates and on agar slides was demonstrated for the first time. This contact deposition technology delivers nanoliter (nL) droplets of bacterial suspensions from a microtiter plate onto a slide surface. Protocols for printing microarrays of whole-cells on agar and on infrared (IR)-transparent slides were evaluated and optimized for subsequent measurement by IR microspectroscopy and IR imaging. Parameters that were investigated included pin capacity, deposition mode, and spatial distribution of microarrays. Bacteria representing eight genera (Yersinia, Staphylococcus, Salmonella, Listeria, Enterobacter, Citrobacter, Klebsiella, and Escherichia) were used in this proof-of-concept study. The resulting dendrograms generated by hierarchical cluster analysis (HCA) demonstrated clustering of the descendants of the foodborne bacteria investigated into their respective branches. The suitability of microarray printing coupled to focal-plane-array (FPA) FTIR detection for the rapid identification of bacteria was demonstrated. Published by Elsevier B.V.
Lipids, 1994
This study reports the structural elucidation of diunsaturated 5-or 6-membered ring cyclic fatty ... more This study reports the structural elucidation of diunsaturated 5-or 6-membered ring cyclic fatty acid monomers (CFAM) isolated from heated flaxseed oil by complementary gas chromatography (GC)-mass spectrometry (MS) and GC-matrix isolation-Fourier transform infrared spectroscopy (MI-FTIR). Infrared measurements of CFAM were carried out on methyl ester derivatives as well-resolved chromatograms were obtained on a polar 100% cyanopropyl polysiloxane capillary GC column. By contrast, electron ionization MS of methyl ester derivatives was of limited value because of double bond migration during the ionization process in the mass spectrometer. This communication reports definitive MS fragmentation patterns that can confirm ring position and double bond position along the fatty acid chain in 1,2-disubstituted CFAM determined as 2-alkenyl-4,4-dimethyloxazoline derivatives. Double bond configuration (cis, trans, or conjugated cis, cis) in CFAM was confirmed by GC-MI-FTIR. The presence of CFAM, degradation products found in used frying oils, is a potential source of dietary toxicity.
Lipids, 1994
This study reports the structural elucidation of diunsaturated 5-or 6-membered ring cyclic fatty ... more This study reports the structural elucidation of diunsaturated 5-or 6-membered ring cyclic fatty acid monomers (CFAM) isolated from heated flaxseed oil by complementary gas chromatography (GC)-mass spectrometry (MS) and GC-matrix isolation-Fourier transform infrared spectroscopy (MI-FTIR). Infrared measurements of CFAM were carried out on methyl ester derivatives as well-resolved chromatograms were obtained on a polar 100% cyanopropyl polysiloxane capillary GC column. By contrast, electron ionization MS of methyl ester derivatives was of limited value because of double bond migration during the ionization process in the mass spectrometer. This communication reports definitive MS fragmentation patterns that can confirm ring position and double bond position along the fatty acid chain in 1,2-disubstituted CFAM determined as 2-alkenyl-4,4-dimethyloxazoline derivatives. Double bond configuration (cis, trans, or conjugated cis, cis) in CFAM was confirmed by GC-MI-FTIR. The presence of CFAM, degradation products found in used frying oils, is a potential source of dietary toxicity.
Lipids, 1995
The objective of this study was to identify oxidation products of conjugated linoleic acid (CLA),... more The objective of this study was to identify oxidation products of conjugated linoleic acid (CLA), a series of octadecadienoic acids with conjugated double bonds, which have been reported to have antioxidant and anticarcinogenic properties. Reference materials of CLA were oxidized in different concentrations of water/methanol; for example, 0.5 g octadecadienoic acid was dissolved in 50 mL methanol, and 100 mL water was added; this suspension was heated at 50 degrees C and continuously aerated. Aliquots of 5 mL were taken over time, extracted with ether, treated with diazomethane and examined by gas chromatography/mass spectrometry and/or gas chromatography with flame-ionization detection. Products identified included the following furan fatty acids (FFAs): 8,11-epoxy-8,10-octadecadienoic; 9,12-epoxy-9,11-octadecadienoic; 10,13-epoxy-10,12-octadecadienoic; and 11,14-epoxy-11,13-octadecadienoic. Conjugated dienes should be considered as a possible source of FFAs, and CLA may have products common to furans in their overall oxidative scheme.
Lipids, 1995
The objective of this study was to identify oxidation products of conjugated linoleic acid (CLA),... more The objective of this study was to identify oxidation products of conjugated linoleic acid (CLA), a series of octadecadienoic acids with conjugated double bonds, which have been reported to have antioxidant and anticarcinogenic properties. Reference materials of CLA were oxidized in different concentrations of water/methanol; for example, 0.5 g octadecadienoic acid was dissolved in 50 mL methanol, and 100 mL water was added; this suspension was heated at 50 degrees C and continuously aerated. Aliquots of 5 mL were taken over time, extracted with ether, treated with diazomethane and examined by gas chromatography/mass spectrometry and/or gas chromatography with flame-ionization detection. Products identified included the following furan fatty acids (FFAs): 8,11-epoxy-8,10-octadecadienoic; 9,12-epoxy-9,11-octadecadienoic; 10,13-epoxy-10,12-octadecadienoic; and 11,14-epoxy-11,13-octadecadienoic. Conjugated dienes should be considered as a possible source of FFAs, and CLA may have products common to furans in their overall oxidative scheme.
Lipids, 1998
Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerizati... more Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerization of sunflower oil, at 2% of the basal diet, from 61.5 to 106 kg live weight, and were compared to pigs fed the same basal diet with 2% added sunflower oil. The total lipids from liver, heart, inner back fat, and omental fat of pigs fed the CLA diet were analyzed for the incorporation of CLA isomers into all the tissue lipid classes. A total of 10 lipid classes were isolated by three-directional thin-layer chromatography and analyzed by gas chromatography (GC) on long capillary columns and by silver-ion highperformance liquid chromatography (Ag + -HPLC); cholesterol was determined spectrophotometrically. Only trace amounts (<0.1%; by GC) of the 9,11-18:2 cis/trans and trans,trans isomers were observed in pigs fed the control diet. Ten and twelve CLA isomers in the diet and in pig tissue lipids were separated by GC and Ag + -HPLC, respectively. The relative concentration of all the CLA isomers in the different lipid classes ranged from 1 to 6% of the total fatty acids. The four major cis/trans isomers (18.9% 11 cis,13 trans-18:2; 26.3% 10 trans,12 cis-18:2; 20.4% 9 cis,11 trans-18:2; and 16.1% 8 trans,10 cis-18:2) constituted 82% of the total CLA isomers in the dietary CLA mixture, and smaller amounts of the corresponding cis,cis (7.4%) and trans,trans (10.1%) isomers were present. The distribution of CLA isomers in inner back fat and in omental fat of the pigs was similar to that found in the diet. The liver triacylglycerols (TAG), free fatty acids (FFA), and cholesteryl esters showed a similar pattern to that found in the diet. The major liver phospholipids showed a marked increase of 9 cis,11 trans-18:2, ranging from 36 to 54%, compared to that present in the diet. However, liver diphosphatidylglycerol (DPG) showed a high incorporation of the 11 cis,13 trans-18:2 isomer (43%). All heart lipid classes, except TAG, showed a high content of 11 cis,13 trans-18:2, which was in marked contrast to results in the liver. The relative proportion of 11 cis,13 trans-18:2 ranged from 30% in the FFA to 77% in DPG. The second major isomer in all heart lipids was 9 cis,11 trans-18:2. In both liver and heart lipids the relative proportions of both 10 trans,12 cis-18:2 and 8 trans,10 cis-18:2 were significantly lower compared to that found in the diet. The FFA in liver and heart showed the highest content of trans,trans isomers (31 to 36%) among all the lipid classes. The preferential accumulation of the 11 cis,13 trans-18:2 into cardiac lipids, and in particular the major phospholipid in the inner mitochondrial membrane, DPG, in both heart and liver, appears unique and may be of concern. The levels of 11 cis,13 trans-18:2 naturally found in foods have not been established.
Lipids, 1998
Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerizati... more Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerization of sunflower oil, at 2% of the basal diet, from 61.5 to 106 kg live weight, and were compared to pigs fed the same basal diet with 2% added sunflower oil. The total lipids from liver, heart, inner back fat, and omental fat of pigs fed the CLA diet were analyzed for the incorporation of CLA isomers into all the tissue lipid classes. A total of 10 lipid classes were isolated by three-directional thin-layer chromatography and analyzed by gas chromatography (GC) on long capillary columns and by silver-ion highperformance liquid chromatography (Ag + -HPLC); cholesterol was determined spectrophotometrically. Only trace amounts (<0.1%; by GC) of the 9,11-18:2 cis/trans and trans,trans isomers were observed in pigs fed the control diet. Ten and twelve CLA isomers in the diet and in pig tissue lipids were separated by GC and Ag + -HPLC, respectively. The relative concentration of all the CLA isomers in the different lipid classes ranged from 1 to 6% of the total fatty acids. The four major cis/trans isomers (18.9% 11 cis,13 trans-18:2; 26.3% 10 trans,12 cis-18:2; 20.4% 9 cis,11 trans-18:2; and 16.1% 8 trans,10 cis-18:2) constituted 82% of the total CLA isomers in the dietary CLA mixture, and smaller amounts of the corresponding cis,cis (7.4%) and trans,trans (10.1%) isomers were present. The distribution of CLA isomers in inner back fat and in omental fat of the pigs was similar to that found in the diet. The liver triacylglycerols (TAG), free fatty acids (FFA), and cholesteryl esters showed a similar pattern to that found in the diet. The major liver phospholipids showed a marked increase of 9 cis,11 trans-18:2, ranging from 36 to 54%, compared to that present in the diet. However, liver diphosphatidylglycerol (DPG) showed a high incorporation of the 11 cis,13 trans-18:2 isomer (43%). All heart lipid classes, except TAG, showed a high content of 11 cis,13 trans-18:2, which was in marked contrast to results in the liver. The relative proportion of 11 cis,13 trans-18:2 ranged from 30% in the FFA to 77% in DPG. The second major isomer in all heart lipids was 9 cis,11 trans-18:2. In both liver and heart lipids the relative proportions of both 10 trans,12 cis-18:2 and 8 trans,10 cis-18:2 were significantly lower compared to that found in the diet. The FFA in liver and heart showed the highest content of trans,trans isomers (31 to 36%) among all the lipid classes. The preferential accumulation of the 11 cis,13 trans-18:2 into cardiac lipids, and in particular the major phospholipid in the inner mitochondrial membrane, DPG, in both heart and liver, appears unique and may be of concern. The levels of 11 cis,13 trans-18:2 naturally found in foods have not been established.
Lipids, 1997
Milk analysis is receiving increased attention. Milk contains conjugated octadecadienoic acids (1... more Milk analysis is receiving increased attention. Milk contains conjugated octadecadienoic acids (18:2) purported to be anticarcinogenic, low levels of essential fatty acids, and trans fatty acids that increase when essential fatty acids are increased in dairy rations. Milk and rumen fatty acid methyl esters (FAME) were prepared using several acid-(HCl, BF 3 , acetyl chloride, H 2 SO 4 ) or base-catalysts (NaOCH 3 , tetramethylguanidine, diazomethane), or combinations thereof. All acid-catalyzed procedures resulted in decreased cis/trans (∆9c,11t-18:2) and increased trans/trans (∆9t,11t-18:2) conjugated dienes and the production of allylic methoxy artifacts. The methoxy artifacts were identified by gas-liquid chromatography (GLC)-mass spectroscopy. The base-catalyzed procedures gave no isomerization of conjugated dienes and no methoxy artifacts, but they did not transesterify N-acyl lipids such as sphingomyelin, and NaOCH 3 did not methylate free fatty acids. In addition, reaction with tetramethylguanidine coextracted material with hexane that interfered with the determination of the short-chain FAME by GLC. Acid-catalyzed methylation resulted in the loss of about 12% total conjugated dienes, 42% recovery of the ∆9c,11t-18:2 isomer, a fourfold increase in ∆9t,11t-18:2, and the formation of methoxy artifacts, compared with the base-catalyzed reactions. Total milk FAME showed significant infrared (IR) absorption due to conjugated dienes at 985 and 948 cm −1 . The IR determination of total trans content of milk FAME was not fully satisfactory because the 966 cm −1 trans band overlapped with the conjugated diene bands. IR accuracy was limited by the fact that the absorptivity of methyl elaidate, used as calibration standard, was different from those of the other minor trans fatty acids (e.g., dienes) found in milk. In addition, acid-catalyzed reactions produced interfering material that absorbed extensively in the trans IR region. No single method or combination of methods could adequately prepare FAME from all lipid classes in milk or rumen lipids, and not affect the conjugated dienes. The best compromise for milk fatty acids was obtained with NaOCH 3 followed by HCl or BF 3 , or diazomethane followed by NaOCH 3 , being aware that sphingomyelins are ig-nored. For rumen samples, the best method was diazomethane followed by NaOCH 3 .
Lipids, 1999
Operating irom one to six silver ion-high-periormance liquid chromatography (Ag·-HPLO columns in ... more Operating irom one to six silver ion-high-periormance liquid chromatography (Ag·-HPLO columns in series progressively improved the resolution of the methvl esters of conjugated linoleic acid (CLA) isomeric mixtures ir~m natural and commercial products. In natural products. the 8 trans. 10 cis-octadecadienoic (18:2) acid was resolved irom the more abundant 7 trans. 9 cis-18:2. and the 10 trans. 12 cis-18:2 was separated irom the major 9 cis. 11 trans-18:2 peak. In addition, both 11 trans. 13 cis-18:2 and 11 cis, 13 trans-18:2 isomers were iound in natural products and were separated: the presence oi the latter. 11 cis, 13 trans-18:2, was established in commercial CLA preparations. Three Ag+-HPLC columns in series appeared to be the best compromise to obtain satisiactorv resolution oi most CLA isomers iound in natural products. A smgle Ag+-HPLC column in series with one oi several normal-phase columns did not improve the resolution oi CLA isomers as compared to that of the iormer alone. The 20:2 conjugated iatty acid isomers 11 cis. 13 trans-20:2 and 12 trans. 14 cis-20:2. which were svnthesized by alkali isomerization irom 11 CIS. 14 ClS-20:2. eluted in the same region oi the Ag·-HPLC chromatogram lust beiore the corresponding geometric CLA isomers. Therefore, CLA Isomers will require Isolation based on cham length prior to Ag--HPLC separation. The positions of conjugated double bonds in 20:2 and 18:2 isomers were established hy gas chromatographY-electron ionization mass spectrometry as their 4.4-dimethvloxazoline derivatives. The double-hand geometry was determined by gas chromatographv-dlrect deposltlon-Fourier transiorm mfrared spectroscopy and hv the Ag' -HPLC relative elution order.
Lipids, 1999
Operating irom one to six silver ion-high-periormance liquid chromatography (Ag·-HPLO columns in ... more Operating irom one to six silver ion-high-periormance liquid chromatography (Ag·-HPLO columns in series progressively improved the resolution of the methvl esters of conjugated linoleic acid (CLA) isomeric mixtures ir~m natural and commercial products. In natural products. the 8 trans. 10 cis-octadecadienoic (18:2) acid was resolved irom the more abundant 7 trans. 9 cis-18:2. and the 10 trans. 12 cis-18:2 was separated irom the major 9 cis. 11 trans-18:2 peak. In addition, both 11 trans. 13 cis-18:2 and 11 cis, 13 trans-18:2 isomers were iound in natural products and were separated: the presence oi the latter. 11 cis, 13 trans-18:2, was established in commercial CLA preparations. Three Ag+-HPLC columns in series appeared to be the best compromise to obtain satisiactorv resolution oi most CLA isomers iound in natural products. A smgle Ag+-HPLC column in series with one oi several normal-phase columns did not improve the resolution oi CLA isomers as compared to that of the iormer alone. The 20:2 conjugated iatty acid isomers 11 cis. 13 trans-20:2 and 12 trans. 14 cis-20:2. which were svnthesized by alkali isomerization irom 11 CIS. 14 ClS-20:2. eluted in the same region oi the Ag·-HPLC chromatogram lust beiore the corresponding geometric CLA isomers. Therefore, CLA Isomers will require Isolation based on cham length prior to Ag--HPLC separation. The positions of conjugated double bonds in 20:2 and 18:2 isomers were established hy gas chromatographY-electron ionization mass spectrometry as their 4.4-dimethvloxazoline derivatives. The double-hand geometry was determined by gas chromatographv-dlrect deposltlon-Fourier transiorm mfrared spectroscopy and hv the Ag' -HPLC relative elution order.
Lipids, 1999
ABSTRAO: Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or 1... more ABSTRAO: Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or 1 2 catalyst. The resultant mixtures of the eight cis/trans geometric isomers of 8, 10-, 9,11-, 10,12-, and 11,13-octadecadienoic (18:2) acid methyl esters were separated by silver ion-high-performance liquid chromatography (Ag+-HPLC) and gas chromatography (GC). Ag+-HPLC allowed the separation of all positional CLA isomers and geometric cis/trans CLA isomers except 10,12-18:2. However, one of the 8,10 isomers (8 cis, 1Otrans-18:2J coeluted with the 9trans,ll cis-18:2 isomer. There were differences in the elution order of the pairs of geometric CLA isomers resolved by Ag+-HPLC. For the 8,10 and 9,11 CLA isomers, cis,trans eluted before trans,cis, whereas the opposite elution pattern was observed for the 11,13-18:2 geometric isomers (trans,cis before cis, trans). All eight cis/trans CLA isomers were separated by GC on long polar capillary columns only when their relative concentrations were about equal. Large differences in the relative concentration of the CLA isomers found in natural products obscured the resolution and identification of a number of minor CLA isomers. In such cases, GC-mass spectrometry of the dimethyloxazoline derivatives was used to identify and confirm coeluting CLA isomers. For the same positional isomer, the cis,trans consistently eluted before the trans,cis CLA isomers by Gc. High resolution mass spectrometry (MS) selected ion recording (SIR) of the molecular ions of the 18:1, 18:2, and 18:3 fatty acid methyl esters served as an independent and highly sensitive method to confirm CLA methyl ester peak assignments in GC chromatograms obtained from food samples by flame-ionization detection. The high-resolution MS data were used to correct for the nonselectivity of the flame-ionization detector.
Lipids, 1999
ABSTRAO: Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or 1... more ABSTRAO: Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or 1 2 catalyst. The resultant mixtures of the eight cis/trans geometric isomers of 8, 10-, 9,11-, 10,12-, and 11,13-octadecadienoic (18:2) acid methyl esters were separated by silver ion-high-performance liquid chromatography (Ag+-HPLC) and gas chromatography (GC). Ag+-HPLC allowed the separation of all positional CLA isomers and geometric cis/trans CLA isomers except 10,12-18:2. However, one of the 8,10 isomers (8 cis, 1Otrans-18:2J coeluted with the 9trans,ll cis-18:2 isomer. There were differences in the elution order of the pairs of geometric CLA isomers resolved by Ag+-HPLC. For the 8,10 and 9,11 CLA isomers, cis,trans eluted before trans,cis, whereas the opposite elution pattern was observed for the 11,13-18:2 geometric isomers (trans,cis before cis, trans). All eight cis/trans CLA isomers were separated by GC on long polar capillary columns only when their relative concentrations were about equal. Large differences in the relative concentration of the CLA isomers found in natural products obscured the resolution and identification of a number of minor CLA isomers. In such cases, GC-mass spectrometry of the dimethyloxazoline derivatives was used to identify and confirm coeluting CLA isomers. For the same positional isomer, the cis,trans consistently eluted before the trans,cis CLA isomers by Gc. High resolution mass spectrometry (MS) selected ion recording (SIR) of the molecular ions of the 18:1, 18:2, and 18:3 fatty acid methyl esters served as an independent and highly sensitive method to confirm CLA methyl ester peak assignments in GC chromatograms obtained from food samples by flame-ionization detection. The high-resolution MS data were used to correct for the nonselectivity of the flame-ionization detector.
Lipids, 1999
The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mi... more The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mixtures of conjugated linoleic acid (CLA) isomers during esterification of mixed CLA free fatty acids and during hydrolysis of mixed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11-18:2. A commercial CLA methyl ester preparation, containing at least 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-11-18:2 methyl ester occurred, with negligible hydrolysis of other CLA isomers. The content of cis-9,trans-11-18:2 in the resulting free fatty acid fraction was between 94 (lipase B reaction) and 77% (GC-4 reaction). The commercial CLA mixture contained only trace amounts of trans-9,cis-11-18:2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analogous results were obtained with these enzymes in the esterification in organic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a methyl ester fraction that contained >98% cis-9,trans-11-18:2. Geotrichum candidum lipases B and GC-4 also demonstrated high selectivity in the esterification of CLA with ethanol, generating ethyl ester fractions containing 96 and 80%, respectively, of the cis-9,trans-11 isomer. In a second set of experiments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was subjected to esterification with octanol in an aqueous reaction system using Amano GC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-11 isomer. After adjustment of the reaction conditions, a concentration of 85% trans-10,cis-12-18:2 could be obtained in the unreacted free fatty acid fraction. These lipase-catalyzed reactions provide a means for the preparative-scale production of high-purity cis-9,trans-11-18:2, and a corresponding CLA fraction depleted of this isomer.
Lipids, 1999
The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mi... more The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mixtures of conjugated linoleic acid (CLA) isomers during esterification of mixed CLA free fatty acids and during hydrolysis of mixed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11-18:2. A commercial CLA methyl ester preparation, containing at least 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-11-18:2 methyl ester occurred, with negligible hydrolysis of other CLA isomers. The content of cis-9,trans-11-18:2 in the resulting free fatty acid fraction was between 94 (lipase B reaction) and 77% (GC-4 reaction). The commercial CLA mixture contained only trace amounts of trans-9,cis-11-18:2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analogous results were obtained with these enzymes in the esterification in organic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a methyl ester fraction that contained >98% cis-9,trans-11-18:2. Geotrichum candidum lipases B and GC-4 also demonstrated high selectivity in the esterification of CLA with ethanol, generating ethyl ester fractions containing 96 and 80%, respectively, of the cis-9,trans-11 isomer. In a second set of experiments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was subjected to esterification with octanol in an aqueous reaction system using Amano GC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-11 isomer. After adjustment of the reaction conditions, a concentration of 85% trans-10,cis-12-18:2 could be obtained in the unreacted free fatty acid fraction. These lipase-catalyzed reactions provide a means for the preparative-scale production of high-purity cis-9,trans-11-18:2, and a corresponding CLA fraction depleted of this isomer.
Journal of the American Chemical Society, 1980
7 8 and 27.12 (t) ppm, indicating that it was the syn-antisyn isomer, 7.1° The I3C NMR spectra of... more 7 8 and 27.12 (t) ppm, indicating that it was the syn-antisyn isomer, 7.1° The I3C NMR spectra of the minor isomer, mp 175-180 C, showed only four absorptions at 136.19 (s), 48.91 (t), 41.05 (d), and 27.09 (t) ppm, indicating that it was the all-syn isomer, 8. The formation of trimers, ...
Lipids, 1998
The identity of a previously unrecognized conjugated linoleic acid (CLA) isomer, 7 trans, 9 cis-o... more The identity of a previously unrecognized conjugated linoleic acid (CLA) isomer, 7 trans, 9 cis-octadecadienoic acid 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, 803-809 (1998).
Journal of The American Oil Chemists Society, 1993
Interest in conjugated-diene fatty acids in foods has recently been increased by discovery of the... more Interest in conjugated-diene fatty acids in foods has recently been increased by discovery of their antioxidant and anticarcinogenic properties. Conjugated octadecatrienes (COTs), members of another group of fatty acids, are also present in foods. COTs are formed during the processing of vegetable oils as the result of the dehydration of secondary oxidation products of linoleic acid. Little information is available
Journal of High Resolution Chromatography, 1999
A commercial mixture of conjugated linoleic acid (CLA) isomers, reportedly consisting of six comp... more A commercial mixture of conjugated linoleic acid (CLA) isomers, reportedly consisting of six components, was recently resolved into 12 peaks attributed to CLA isomers using silver-ion high performance liquid chromatography (Ag + -HPLC). In this study, the coupling of two ...
Journal of High Resolution Chromatography, 1999
A commercial mixture of conjugated linoleic acid (CLA) isomers, reportedly consisting of six comp... more A commercial mixture of conjugated linoleic acid (CLA) isomers, reportedly consisting of six components, was recently resolved into 12 peaks attributed to CLA isomers using silver-ion high performance liquid chromatography (Ag + -HPLC). In this study, the coupling of two ...
Vibrational Spectroscopy, 2005
Fourier transform infrared (FTIR) spectroscopy has been established as a rapid bacteria identific... more Fourier transform infrared (FTIR) spectroscopy has been established as a rapid bacteria identification technique. To increase the throughput of this methodology, the feasibility of applying contact deposition microarray technology to print intact bacterial cells as arrayed deposits (150 mm diameter) on optical substrates and on agar slides was demonstrated for the first time. This contact deposition technology delivers nanoliter (nL) droplets of bacterial suspensions from a microtiter plate onto a slide surface. Protocols for printing microarrays of whole-cells on agar and on infrared (IR)-transparent slides were evaluated and optimized for subsequent measurement by IR microspectroscopy and IR imaging. Parameters that were investigated included pin capacity, deposition mode, and spatial distribution of microarrays. Bacteria representing eight genera (Yersinia, Staphylococcus, Salmonella, Listeria, Enterobacter, Citrobacter, Klebsiella, and Escherichia) were used in this proof-of-concept study. The resulting dendrograms generated by hierarchical cluster analysis (HCA) demonstrated clustering of the descendants of the foodborne bacteria investigated into their respective branches. The suitability of microarray printing coupled to focal-plane-array (FPA) FTIR detection for the rapid identification of bacteria was demonstrated. Published by Elsevier B.V.
Lipids, 1994
This study reports the structural elucidation of diunsaturated 5-or 6-membered ring cyclic fatty ... more This study reports the structural elucidation of diunsaturated 5-or 6-membered ring cyclic fatty acid monomers (CFAM) isolated from heated flaxseed oil by complementary gas chromatography (GC)-mass spectrometry (MS) and GC-matrix isolation-Fourier transform infrared spectroscopy (MI-FTIR). Infrared measurements of CFAM were carried out on methyl ester derivatives as well-resolved chromatograms were obtained on a polar 100% cyanopropyl polysiloxane capillary GC column. By contrast, electron ionization MS of methyl ester derivatives was of limited value because of double bond migration during the ionization process in the mass spectrometer. This communication reports definitive MS fragmentation patterns that can confirm ring position and double bond position along the fatty acid chain in 1,2-disubstituted CFAM determined as 2-alkenyl-4,4-dimethyloxazoline derivatives. Double bond configuration (cis, trans, or conjugated cis, cis) in CFAM was confirmed by GC-MI-FTIR. The presence of CFAM, degradation products found in used frying oils, is a potential source of dietary toxicity.
Lipids, 1994
This study reports the structural elucidation of diunsaturated 5-or 6-membered ring cyclic fatty ... more This study reports the structural elucidation of diunsaturated 5-or 6-membered ring cyclic fatty acid monomers (CFAM) isolated from heated flaxseed oil by complementary gas chromatography (GC)-mass spectrometry (MS) and GC-matrix isolation-Fourier transform infrared spectroscopy (MI-FTIR). Infrared measurements of CFAM were carried out on methyl ester derivatives as well-resolved chromatograms were obtained on a polar 100% cyanopropyl polysiloxane capillary GC column. By contrast, electron ionization MS of methyl ester derivatives was of limited value because of double bond migration during the ionization process in the mass spectrometer. This communication reports definitive MS fragmentation patterns that can confirm ring position and double bond position along the fatty acid chain in 1,2-disubstituted CFAM determined as 2-alkenyl-4,4-dimethyloxazoline derivatives. Double bond configuration (cis, trans, or conjugated cis, cis) in CFAM was confirmed by GC-MI-FTIR. The presence of CFAM, degradation products found in used frying oils, is a potential source of dietary toxicity.
Lipids, 1995
The objective of this study was to identify oxidation products of conjugated linoleic acid (CLA),... more The objective of this study was to identify oxidation products of conjugated linoleic acid (CLA), a series of octadecadienoic acids with conjugated double bonds, which have been reported to have antioxidant and anticarcinogenic properties. Reference materials of CLA were oxidized in different concentrations of water/methanol; for example, 0.5 g octadecadienoic acid was dissolved in 50 mL methanol, and 100 mL water was added; this suspension was heated at 50 degrees C and continuously aerated. Aliquots of 5 mL were taken over time, extracted with ether, treated with diazomethane and examined by gas chromatography/mass spectrometry and/or gas chromatography with flame-ionization detection. Products identified included the following furan fatty acids (FFAs): 8,11-epoxy-8,10-octadecadienoic; 9,12-epoxy-9,11-octadecadienoic; 10,13-epoxy-10,12-octadecadienoic; and 11,14-epoxy-11,13-octadecadienoic. Conjugated dienes should be considered as a possible source of FFAs, and CLA may have products common to furans in their overall oxidative scheme.
Lipids, 1995
The objective of this study was to identify oxidation products of conjugated linoleic acid (CLA),... more The objective of this study was to identify oxidation products of conjugated linoleic acid (CLA), a series of octadecadienoic acids with conjugated double bonds, which have been reported to have antioxidant and anticarcinogenic properties. Reference materials of CLA were oxidized in different concentrations of water/methanol; for example, 0.5 g octadecadienoic acid was dissolved in 50 mL methanol, and 100 mL water was added; this suspension was heated at 50 degrees C and continuously aerated. Aliquots of 5 mL were taken over time, extracted with ether, treated with diazomethane and examined by gas chromatography/mass spectrometry and/or gas chromatography with flame-ionization detection. Products identified included the following furan fatty acids (FFAs): 8,11-epoxy-8,10-octadecadienoic; 9,12-epoxy-9,11-octadecadienoic; 10,13-epoxy-10,12-octadecadienoic; and 11,14-epoxy-11,13-octadecadienoic. Conjugated dienes should be considered as a possible source of FFAs, and CLA may have products common to furans in their overall oxidative scheme.
Lipids, 1998
Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerizati... more Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerization of sunflower oil, at 2% of the basal diet, from 61.5 to 106 kg live weight, and were compared to pigs fed the same basal diet with 2% added sunflower oil. The total lipids from liver, heart, inner back fat, and omental fat of pigs fed the CLA diet were analyzed for the incorporation of CLA isomers into all the tissue lipid classes. A total of 10 lipid classes were isolated by three-directional thin-layer chromatography and analyzed by gas chromatography (GC) on long capillary columns and by silver-ion highperformance liquid chromatography (Ag + -HPLC); cholesterol was determined spectrophotometrically. Only trace amounts (<0.1%; by GC) of the 9,11-18:2 cis/trans and trans,trans isomers were observed in pigs fed the control diet. Ten and twelve CLA isomers in the diet and in pig tissue lipids were separated by GC and Ag + -HPLC, respectively. The relative concentration of all the CLA isomers in the different lipid classes ranged from 1 to 6% of the total fatty acids. The four major cis/trans isomers (18.9% 11 cis,13 trans-18:2; 26.3% 10 trans,12 cis-18:2; 20.4% 9 cis,11 trans-18:2; and 16.1% 8 trans,10 cis-18:2) constituted 82% of the total CLA isomers in the dietary CLA mixture, and smaller amounts of the corresponding cis,cis (7.4%) and trans,trans (10.1%) isomers were present. The distribution of CLA isomers in inner back fat and in omental fat of the pigs was similar to that found in the diet. The liver triacylglycerols (TAG), free fatty acids (FFA), and cholesteryl esters showed a similar pattern to that found in the diet. The major liver phospholipids showed a marked increase of 9 cis,11 trans-18:2, ranging from 36 to 54%, compared to that present in the diet. However, liver diphosphatidylglycerol (DPG) showed a high incorporation of the 11 cis,13 trans-18:2 isomer (43%). All heart lipid classes, except TAG, showed a high content of 11 cis,13 trans-18:2, which was in marked contrast to results in the liver. The relative proportion of 11 cis,13 trans-18:2 ranged from 30% in the FFA to 77% in DPG. The second major isomer in all heart lipids was 9 cis,11 trans-18:2. In both liver and heart lipids the relative proportions of both 10 trans,12 cis-18:2 and 8 trans,10 cis-18:2 were significantly lower compared to that found in the diet. The FFA in liver and heart showed the highest content of trans,trans isomers (31 to 36%) among all the lipid classes. The preferential accumulation of the 11 cis,13 trans-18:2 into cardiac lipids, and in particular the major phospholipid in the inner mitochondrial membrane, DPG, in both heart and liver, appears unique and may be of concern. The levels of 11 cis,13 trans-18:2 naturally found in foods have not been established.
Lipids, 1998
Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerizati... more Pigs were fed a commercial conjugated linoleic acid (CLA) mixture, prepared by alkali isomerization of sunflower oil, at 2% of the basal diet, from 61.5 to 106 kg live weight, and were compared to pigs fed the same basal diet with 2% added sunflower oil. The total lipids from liver, heart, inner back fat, and omental fat of pigs fed the CLA diet were analyzed for the incorporation of CLA isomers into all the tissue lipid classes. A total of 10 lipid classes were isolated by three-directional thin-layer chromatography and analyzed by gas chromatography (GC) on long capillary columns and by silver-ion highperformance liquid chromatography (Ag + -HPLC); cholesterol was determined spectrophotometrically. Only trace amounts (<0.1%; by GC) of the 9,11-18:2 cis/trans and trans,trans isomers were observed in pigs fed the control diet. Ten and twelve CLA isomers in the diet and in pig tissue lipids were separated by GC and Ag + -HPLC, respectively. The relative concentration of all the CLA isomers in the different lipid classes ranged from 1 to 6% of the total fatty acids. The four major cis/trans isomers (18.9% 11 cis,13 trans-18:2; 26.3% 10 trans,12 cis-18:2; 20.4% 9 cis,11 trans-18:2; and 16.1% 8 trans,10 cis-18:2) constituted 82% of the total CLA isomers in the dietary CLA mixture, and smaller amounts of the corresponding cis,cis (7.4%) and trans,trans (10.1%) isomers were present. The distribution of CLA isomers in inner back fat and in omental fat of the pigs was similar to that found in the diet. The liver triacylglycerols (TAG), free fatty acids (FFA), and cholesteryl esters showed a similar pattern to that found in the diet. The major liver phospholipids showed a marked increase of 9 cis,11 trans-18:2, ranging from 36 to 54%, compared to that present in the diet. However, liver diphosphatidylglycerol (DPG) showed a high incorporation of the 11 cis,13 trans-18:2 isomer (43%). All heart lipid classes, except TAG, showed a high content of 11 cis,13 trans-18:2, which was in marked contrast to results in the liver. The relative proportion of 11 cis,13 trans-18:2 ranged from 30% in the FFA to 77% in DPG. The second major isomer in all heart lipids was 9 cis,11 trans-18:2. In both liver and heart lipids the relative proportions of both 10 trans,12 cis-18:2 and 8 trans,10 cis-18:2 were significantly lower compared to that found in the diet. The FFA in liver and heart showed the highest content of trans,trans isomers (31 to 36%) among all the lipid classes. The preferential accumulation of the 11 cis,13 trans-18:2 into cardiac lipids, and in particular the major phospholipid in the inner mitochondrial membrane, DPG, in both heart and liver, appears unique and may be of concern. The levels of 11 cis,13 trans-18:2 naturally found in foods have not been established.
Lipids, 1997
Milk analysis is receiving increased attention. Milk contains conjugated octadecadienoic acids (1... more Milk analysis is receiving increased attention. Milk contains conjugated octadecadienoic acids (18:2) purported to be anticarcinogenic, low levels of essential fatty acids, and trans fatty acids that increase when essential fatty acids are increased in dairy rations. Milk and rumen fatty acid methyl esters (FAME) were prepared using several acid-(HCl, BF 3 , acetyl chloride, H 2 SO 4 ) or base-catalysts (NaOCH 3 , tetramethylguanidine, diazomethane), or combinations thereof. All acid-catalyzed procedures resulted in decreased cis/trans (∆9c,11t-18:2) and increased trans/trans (∆9t,11t-18:2) conjugated dienes and the production of allylic methoxy artifacts. The methoxy artifacts were identified by gas-liquid chromatography (GLC)-mass spectroscopy. The base-catalyzed procedures gave no isomerization of conjugated dienes and no methoxy artifacts, but they did not transesterify N-acyl lipids such as sphingomyelin, and NaOCH 3 did not methylate free fatty acids. In addition, reaction with tetramethylguanidine coextracted material with hexane that interfered with the determination of the short-chain FAME by GLC. Acid-catalyzed methylation resulted in the loss of about 12% total conjugated dienes, 42% recovery of the ∆9c,11t-18:2 isomer, a fourfold increase in ∆9t,11t-18:2, and the formation of methoxy artifacts, compared with the base-catalyzed reactions. Total milk FAME showed significant infrared (IR) absorption due to conjugated dienes at 985 and 948 cm −1 . The IR determination of total trans content of milk FAME was not fully satisfactory because the 966 cm −1 trans band overlapped with the conjugated diene bands. IR accuracy was limited by the fact that the absorptivity of methyl elaidate, used as calibration standard, was different from those of the other minor trans fatty acids (e.g., dienes) found in milk. In addition, acid-catalyzed reactions produced interfering material that absorbed extensively in the trans IR region. No single method or combination of methods could adequately prepare FAME from all lipid classes in milk or rumen lipids, and not affect the conjugated dienes. The best compromise for milk fatty acids was obtained with NaOCH 3 followed by HCl or BF 3 , or diazomethane followed by NaOCH 3 , being aware that sphingomyelins are ig-nored. For rumen samples, the best method was diazomethane followed by NaOCH 3 .
Lipids, 1999
Operating irom one to six silver ion-high-periormance liquid chromatography (Ag·-HPLO columns in ... more Operating irom one to six silver ion-high-periormance liquid chromatography (Ag·-HPLO columns in series progressively improved the resolution of the methvl esters of conjugated linoleic acid (CLA) isomeric mixtures ir~m natural and commercial products. In natural products. the 8 trans. 10 cis-octadecadienoic (18:2) acid was resolved irom the more abundant 7 trans. 9 cis-18:2. and the 10 trans. 12 cis-18:2 was separated irom the major 9 cis. 11 trans-18:2 peak. In addition, both 11 trans. 13 cis-18:2 and 11 cis, 13 trans-18:2 isomers were iound in natural products and were separated: the presence oi the latter. 11 cis, 13 trans-18:2, was established in commercial CLA preparations. Three Ag+-HPLC columns in series appeared to be the best compromise to obtain satisiactorv resolution oi most CLA isomers iound in natural products. A smgle Ag+-HPLC column in series with one oi several normal-phase columns did not improve the resolution oi CLA isomers as compared to that of the iormer alone. The 20:2 conjugated iatty acid isomers 11 cis. 13 trans-20:2 and 12 trans. 14 cis-20:2. which were svnthesized by alkali isomerization irom 11 CIS. 14 ClS-20:2. eluted in the same region oi the Ag·-HPLC chromatogram lust beiore the corresponding geometric CLA isomers. Therefore, CLA Isomers will require Isolation based on cham length prior to Ag--HPLC separation. The positions of conjugated double bonds in 20:2 and 18:2 isomers were established hy gas chromatographY-electron ionization mass spectrometry as their 4.4-dimethvloxazoline derivatives. The double-hand geometry was determined by gas chromatographv-dlrect deposltlon-Fourier transiorm mfrared spectroscopy and hv the Ag' -HPLC relative elution order.
Lipids, 1999
Operating irom one to six silver ion-high-periormance liquid chromatography (Ag·-HPLO columns in ... more Operating irom one to six silver ion-high-periormance liquid chromatography (Ag·-HPLO columns in series progressively improved the resolution of the methvl esters of conjugated linoleic acid (CLA) isomeric mixtures ir~m natural and commercial products. In natural products. the 8 trans. 10 cis-octadecadienoic (18:2) acid was resolved irom the more abundant 7 trans. 9 cis-18:2. and the 10 trans. 12 cis-18:2 was separated irom the major 9 cis. 11 trans-18:2 peak. In addition, both 11 trans. 13 cis-18:2 and 11 cis, 13 trans-18:2 isomers were iound in natural products and were separated: the presence oi the latter. 11 cis, 13 trans-18:2, was established in commercial CLA preparations. Three Ag+-HPLC columns in series appeared to be the best compromise to obtain satisiactorv resolution oi most CLA isomers iound in natural products. A smgle Ag+-HPLC column in series with one oi several normal-phase columns did not improve the resolution oi CLA isomers as compared to that of the iormer alone. The 20:2 conjugated iatty acid isomers 11 cis. 13 trans-20:2 and 12 trans. 14 cis-20:2. which were svnthesized by alkali isomerization irom 11 CIS. 14 ClS-20:2. eluted in the same region oi the Ag·-HPLC chromatogram lust beiore the corresponding geometric CLA isomers. Therefore, CLA Isomers will require Isolation based on cham length prior to Ag--HPLC separation. The positions of conjugated double bonds in 20:2 and 18:2 isomers were established hy gas chromatographY-electron ionization mass spectrometry as their 4.4-dimethvloxazoline derivatives. The double-hand geometry was determined by gas chromatographv-dlrect deposltlon-Fourier transiorm mfrared spectroscopy and hv the Ag' -HPLC relative elution order.
Lipids, 1999
ABSTRAO: Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or 1... more ABSTRAO: Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or 1 2 catalyst. The resultant mixtures of the eight cis/trans geometric isomers of 8, 10-, 9,11-, 10,12-, and 11,13-octadecadienoic (18:2) acid methyl esters were separated by silver ion-high-performance liquid chromatography (Ag+-HPLC) and gas chromatography (GC). Ag+-HPLC allowed the separation of all positional CLA isomers and geometric cis/trans CLA isomers except 10,12-18:2. However, one of the 8,10 isomers (8 cis, 1Otrans-18:2J coeluted with the 9trans,ll cis-18:2 isomer. There were differences in the elution order of the pairs of geometric CLA isomers resolved by Ag+-HPLC. For the 8,10 and 9,11 CLA isomers, cis,trans eluted before trans,cis, whereas the opposite elution pattern was observed for the 11,13-18:2 geometric isomers (trans,cis before cis, trans). All eight cis/trans CLA isomers were separated by GC on long polar capillary columns only when their relative concentrations were about equal. Large differences in the relative concentration of the CLA isomers found in natural products obscured the resolution and identification of a number of minor CLA isomers. In such cases, GC-mass spectrometry of the dimethyloxazoline derivatives was used to identify and confirm coeluting CLA isomers. For the same positional isomer, the cis,trans consistently eluted before the trans,cis CLA isomers by Gc. High resolution mass spectrometry (MS) selected ion recording (SIR) of the molecular ions of the 18:1, 18:2, and 18:3 fatty acid methyl esters served as an independent and highly sensitive method to confirm CLA methyl ester peak assignments in GC chromatograms obtained from food samples by flame-ionization detection. The high-resolution MS data were used to correct for the nonselectivity of the flame-ionization detector.
Lipids, 1999
ABSTRAO: Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or 1... more ABSTRAO: Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or 1 2 catalyst. The resultant mixtures of the eight cis/trans geometric isomers of 8, 10-, 9,11-, 10,12-, and 11,13-octadecadienoic (18:2) acid methyl esters were separated by silver ion-high-performance liquid chromatography (Ag+-HPLC) and gas chromatography (GC). Ag+-HPLC allowed the separation of all positional CLA isomers and geometric cis/trans CLA isomers except 10,12-18:2. However, one of the 8,10 isomers (8 cis, 1Otrans-18:2J coeluted with the 9trans,ll cis-18:2 isomer. There were differences in the elution order of the pairs of geometric CLA isomers resolved by Ag+-HPLC. For the 8,10 and 9,11 CLA isomers, cis,trans eluted before trans,cis, whereas the opposite elution pattern was observed for the 11,13-18:2 geometric isomers (trans,cis before cis, trans). All eight cis/trans CLA isomers were separated by GC on long polar capillary columns only when their relative concentrations were about equal. Large differences in the relative concentration of the CLA isomers found in natural products obscured the resolution and identification of a number of minor CLA isomers. In such cases, GC-mass spectrometry of the dimethyloxazoline derivatives was used to identify and confirm coeluting CLA isomers. For the same positional isomer, the cis,trans consistently eluted before the trans,cis CLA isomers by Gc. High resolution mass spectrometry (MS) selected ion recording (SIR) of the molecular ions of the 18:1, 18:2, and 18:3 fatty acid methyl esters served as an independent and highly sensitive method to confirm CLA methyl ester peak assignments in GC chromatograms obtained from food samples by flame-ionization detection. The high-resolution MS data were used to correct for the nonselectivity of the flame-ionization detector.
Lipids, 1999
The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mi... more The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mixtures of conjugated linoleic acid (CLA) isomers during esterification of mixed CLA free fatty acids and during hydrolysis of mixed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11-18:2. A commercial CLA methyl ester preparation, containing at least 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-11-18:2 methyl ester occurred, with negligible hydrolysis of other CLA isomers. The content of cis-9,trans-11-18:2 in the resulting free fatty acid fraction was between 94 (lipase B reaction) and 77% (GC-4 reaction). The commercial CLA mixture contained only trace amounts of trans-9,cis-11-18:2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analogous results were obtained with these enzymes in the esterification in organic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a methyl ester fraction that contained >98% cis-9,trans-11-18:2. Geotrichum candidum lipases B and GC-4 also demonstrated high selectivity in the esterification of CLA with ethanol, generating ethyl ester fractions containing 96 and 80%, respectively, of the cis-9,trans-11 isomer. In a second set of experiments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was subjected to esterification with octanol in an aqueous reaction system using Amano GC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-11 isomer. After adjustment of the reaction conditions, a concentration of 85% trans-10,cis-12-18:2 could be obtained in the unreacted free fatty acid fraction. These lipase-catalyzed reactions provide a means for the preparative-scale production of high-purity cis-9,trans-11-18:2, and a corresponding CLA fraction depleted of this isomer.
Lipids, 1999
The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mi... more The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mixtures of conjugated linoleic acid (CLA) isomers during esterification of mixed CLA free fatty acids and during hydrolysis of mixed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11-18:2. A commercial CLA methyl ester preparation, containing at least 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-11-18:2 methyl ester occurred, with negligible hydrolysis of other CLA isomers. The content of cis-9,trans-11-18:2 in the resulting free fatty acid fraction was between 94 (lipase B reaction) and 77% (GC-4 reaction). The commercial CLA mixture contained only trace amounts of trans-9,cis-11-18:2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analogous results were obtained with these enzymes in the esterification in organic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a methyl ester fraction that contained >98% cis-9,trans-11-18:2. Geotrichum candidum lipases B and GC-4 also demonstrated high selectivity in the esterification of CLA with ethanol, generating ethyl ester fractions containing 96 and 80%, respectively, of the cis-9,trans-11 isomer. In a second set of experiments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was subjected to esterification with octanol in an aqueous reaction system using Amano GC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-11 isomer. After adjustment of the reaction conditions, a concentration of 85% trans-10,cis-12-18:2 could be obtained in the unreacted free fatty acid fraction. These lipase-catalyzed reactions provide a means for the preparative-scale production of high-purity cis-9,trans-11-18:2, and a corresponding CLA fraction depleted of this isomer.
Journal of the American Chemical Society, 1980
7 8 and 27.12 (t) ppm, indicating that it was the syn-antisyn isomer, 7.1° The I3C NMR spectra of... more 7 8 and 27.12 (t) ppm, indicating that it was the syn-antisyn isomer, 7.1° The I3C NMR spectra of the minor isomer, mp 175-180 C, showed only four absorptions at 136.19 (s), 48.91 (t), 41.05 (d), and 27.09 (t) ppm, indicating that it was the all-syn isomer, 8. The formation of trimers, ...
Lipids, 1998
The identity of a previously unrecognized conjugated linoleic acid (CLA) isomer, 7 trans, 9 cis-o... more The identity of a previously unrecognized conjugated linoleic acid (CLA) isomer, 7 trans, 9 cis-octadecadienoic acid 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, 803-809 (1998).